Targeted immunotolerance

ABSTRACT

Methods and compounds for conferring site-specific or local immune privilege.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.62/510,586, filed May 24, 2017, U.S. Provisional Application No.62/510,816, filed May 25, 2017, U.S. Provisional Application No.62/558,175, filed Sep. 13, 2017, and U.S. Provisional Application No.62/595,352, filed Dec. 6, 2017, each of which is hereby incorporated byreference in its entirety.

FIELD

The embodiments provided herein relate to, for example, methods andcompositions for local or targeted immune-privilege.

BACKGROUND

Instances of unwanted immune responses, e.g., as in the rejection oftransplanted tissue or in autoimmune disorders, constitute a majorhealth problem for millions of people across the world. Long-termoutcomes for organ transplantation are frequently characterized bychronic rejection, and eventual failure of the transplanted organ. Morethan twenty autoimmune disorders are known, affecting essentially everyorgan of the body, and affecting over fifty million people in NorthAmerica alone. The broadly active immunosuppressive medications used tocombat the pathogenic immune response in both scenarios have seriousside effects.

SUMMARY

Disclosed herein are methods and therapeutic compounds that providesite-specific immune privilege. Embodiments disclosed herein areincorporated by reference into this section.

In some embodiments, the therapeutic compound comprises an engineeredmulti-specific compound, e.g., an engineered bi-specific molecule, e.g.,an engineered bi-specific antibody molecule, comprising:

1) a specific targeting moiety selected from:

a) a donor specific targeting moiety which, e.g., preferentially binds adonor target (preferentially as compared with binding to a recipientantigen), and is useful for providing site-specific immune privilege fora transplant tissue, e.g., an organ, from a donor; or

b) a tissue specific targeting moiety which, e.g., preferentially bindsa subject target tissue (preferentially as compared with subjectnon-target tissue), and is useful for providing site-specific immuneprivilege for a subject tissue undergoing unwanted immune attack, e.g.,in an autoimmune disorder); and

2) an effector binding/modulating moiety selected from:

(a) an immune cell inhibitory molecule binding/modulating moiety(referred to herein as an ICIM binding/modulating moiety);

(b) an immunosuppressive immune cell binding/modulating moiety (referredto herein as an IIC binding/modulating moiety);

(c) an effector binding/modulating moiety that, as part of a therapeuticcompound, promotes an immuno-suppressive local microenvironment, e.g.,by providing in the proximity of the target, a substance that inhibitsor minimizes attack by the immune system of the target (referred toherein as an SM binding/modulating moiety); or

(d) an immune cell stimulatory molecule binding/modulating moiety(referred to herein as an ICSM binding/modulating moiety), wherein theICSM inhibits immune activation by, for example, blocking theinteraction between a costimulatory molecule and its counterstructure.

An effector binding/modulating moiety can fall into more than one ofclasses a, b and c. E.g., as is shown below, a CTLA4 binding moleculefalls into both of categories a and b.

In some embodiments, the therapeutic compound comprises an ICIMbinding/modulating moiety. In some embodiments, an ICIMbinding/modulating molecule and binds, and agonizes, an inhibitorymolecule, e.g., an inhibitory immune checkpoint molecule, or otherwiseinhibits or reduces the activity of an immune cell, e.g., a cytotoxic Tcell, a B cell, NK cell, or a myeloid cell, e.g., a neutrophil ormacrophage.

In some embodiments, the therapeutic compound comprises an engineeredmulti-specific compound, e.g., an engineered bi-specific molecule, e.g.,an engineered bi-specific antibody molecule, comprising:

1) a specific targeting moiety, e.g., a donor specific targeting moiety(which binds a donor target and is useful for providing site-specificimmune privilege for a transplant tissue, e.g., an organ, from a donor)or a tissue specific targeting moiety (which binds a subject tissuetarget and is useful for providing site-specific immune privilege for asubject tissue undergoing unwanted immune attack, e.g., in an autoimmunedisorder); and

2) an effector binding/modulating moiety comprising an ICIMbinding/modulating moiety that binds to an effector molecule on animmune cell, e.g., an inhibitory receptor, e.g., PD-1, wherein, uponbinding of the specific targeting moiety to its target, and binding ofthe ICIM binding/modulating moiety to an effector molecule on the immunecell, an immune cell activity, e.g., the ability of the immune cell tomount an immune attack, is down regulated, e.g., through an inhibitorysignal dependent on the clustering of effector molecules on the immunecell. In some embodiments, the engineered multi-specific compoundcomprises additional binding moieties so that it binds more than twospecific molecules, such as, but not limited to, 3 or 4.

In some embodiments, the therapeutic compound comprises an ICIMbinding/modulating moiety and has one or both of the followingproperties: (a) the level of down regulation of an immune cell isgreater when the therapeutic compound is bound to its target than whenthe therapeutic compound is not bound to its target; and (b) thetherapeutic compound, when engaged with a cell surface inhibitoryreceptor, e.g., PD-1, on an immune cell, does not inhibit, or does notsubstantially inhibit the ability of the cell surface inhibitoryreceptor to bind an endogenous ligand.

In some embodiments, the level of down regulation of an immune cell isgreater when the therapeutic compound is bound to its target than whenthe therapeutic compound is not bound to its target. In embodiments, thelevel of down regulation by target bound therapeutic compound is equalto or greater than 1.5-fold, 2-fold, 4-fold, 8-fold or 10-fold greaterthan what is seen when it is not bound to its target. In embodiments,therapeutic compound does not, or does not significantly down regulateimmune cells when it is not bound to target. Thus, indiscriminant orunwanted agonism of an inhibitory receptor, e.g., PD-1, is minimized oreliminated. E.g., when the therapeutic compound is bound to an immunecell, but not bound to the targeted moiety, engagement of a inhibitoryimmune checkpoint molecule by the therapeutic compound does not resultin down regulation or does not result in substantia down regulation,e.g., the inhibitory receptor on the immune cell to which thetherapeutic compound is bound, is not clustered or not clusteredsufficiently to result in an inhibitory signal sufficient to give downregulation or substantial inhibition of the immune cell.

In embodiments, the therapeutic compound, when engaged with a cellsurface inhibitory receptor, e.g., PD-1, on an immune cell, does notinhibit, or does not substantially inhibit the ability of the cellsurface inhibitory receptor to bind an endogenous ligand. In someembodiments, the therapeutic compound can bind to the PD-L1/2 bindingsite on PD-1. Thus, indiscriminant or unwanted antagonism of aninhibitory receptor, e.g., PD-1, is minimized or eliminated. Inembodiments, binding of the therapeutic compound to an inhibitoryreceptor, e.g. PD-1, on an immune cell does not impede, or substantiallyimpede, the ability of the inhibitory receptor to bind a natural ligand,e.g., PD-L1. In embodiments, binding of the therapeutic compound to aninhibitory receptor, e.g. PD-1, on an immune reduces binding of anatural ligand, e.g., PD-L1, by less than 50, 40, 30, 20, 10, or 5% ofwhat is seen in the absence of therapeutic compound.

In some embodiments, the therapeutic compound comprises an ICIMbinding/modulating moiety and, when administered to a subject at atherapeutically effective dose, does not result in unacceptable levelsof systemic immune suppression, as would be possible if indiscriminantagonism of the inhibitory receptor in all immune cells of a type, e.g.,all T cells, occurred, or unacceptable levels of systemic immuneactivation, as would be possible if the therapeutic compound antagonizedthe interaction of the inhibitory receptor with its natural ligand.

While not wishing to be bound by theory, it is believed that, uponadministration to a subject, a therapeutic compound comprising an ICIMbinding/modulating moiety can exist in any one of four states: i)unbound and in free solution; ii) bound to only an inhibitory receptorexpressed on the surface of an immune cell, e.g., a T cell, through theICIM binding/modulating moiety; iii) bound to only the surface of thetarget transplant or subject tissue through the targeting moiety; andiv) bound to both the surface of target transplant or subject tissuethrough the targeting moiety and to an inhibitory receptor expressed byan immune cell, e.g., a T cell, through the ICIM binding/modulatingmoiety. When the therapeutic compound is bound only to the targettransplant or subject tissue (iii) through the targeting moiety, it hasno, or no substantial, effect on the target transplant or tissue. Whenthe therapeutic compound is bound to the target transplant or tissuethrough the targeting moiety and bound to an inhibitory receptorexpressed by an immune cell, e.g., a T cell, through the ICIMbinding/modulating moiety (iv), it creates immune privilege at thetarget organ or tissue. While not wishing to be bound by theory, isbelieved that this is achieved by the target transplant or donor tissuemultimerizing the therapeutic compound molecules on its surface, e.g.,by immobilizing a plurality of therapeutic compound molecules at a highdensity and valency. The multimerization of the therapeutic compoundmolecules allows the ICIM binding/modulating moieties of the therapeuticcompounds to promote clustering of inhibitory receptors expressed on thesurface of the immune cell, e.g., a pathogenic T cell, and transmissionof an inhibitory signal functioning to silence or down-regulate theimmune cell. E.g., in the case of T cells, a therapeutic compoundcomprising an ICIM binding/modulating moiety comprising a PD-L1molecule, or an anti-PD-1 Ab, can be used. Binding of a plurality of thetherapeutic compound molecules to the target results in multimerizationof the therapeutic compound molecules, which in turn, by virtue of thePD-L1 molecule, or a functional anti-PD-1 antibody molecule, leads toclustering of PD-1 on the T cell. If that clustering occurs in thecontext of antigen presentation by the target MHC, to T cell receptor onthe T cell, a negative signal is generated and the T cell will beinactivated. In embodiments the ICIM binding/modulating moiety, e.g., afunctional antibody molecule, binds the effector molecule but does notinhibit, or substantially inhibit, interaction of the effector moleculewith its native ligand(s).

In some embodiments, the therapeutic compound comprises an IICbinding/modulating moiety, which binds and recruits an immunesuppressive immune cell, e.g., a Treg, e.g., a Foxp3+CD25+ Treg, to theproximity of the target tissue.

In some embodiments, the therapeutic compound comprises a SMbinding/modulating moiety, which modulates, e.g., binds and inhibits,sequesters, degrades or otherwise neutralizes a substance, e.g., asoluble molecule that modulates an immune response, e.g., ATP or AMP.

In some embodiments, the therapeutic compound comprises a targetingmoiety that is specific for a target on an immune cell. In someembodiments, the target is as described herein. In some embodiments, thetarget is MAdCAM. In some embodiments, the targeting moiety is anantibody that binds to MAdCAM.

In some embodiments the therapeutic compound comprises an ICSMbinding/modulating moiety, which binds a stimulatory molecule, e.g., acostimulatory molecule. In some embodiments, the ICSM inhibits thecostimulatory molecule counterstructure by. Binding/modulating eitherthe costimulatory molecule or the costimulatory moleculecounterstructure can serve to down regulate the ability of an immunecell to mount an immune response. In some embodiments, the ICSMbinding/modulating moiety can bind a stimulatory, e.g., costimulatorymolecule on an immune cell, e.g., OX40 on T cells, or the counter memberof the stimulatory molecule e.g. OX40L on another cell, such as, but notlimited to, immune cells such as NK cells, mast cells, dendritic cells,or, for example, non-immune cells such as endothelial cells, or smoothmuscle cells.

In some embodiments, the therapeutic compound comprises a donor specifictargeting moiety and provides site-specific immune privilege for donortransplant tissue implanted in a subject. In some embodiments, thetherapeutic compound comprises a tissue specific targeting moiety andprovides site-specific immune privilege for a tissue of a subject, e.g.,a tissue afflicted with an unwanted immune response in an autoimmunedisorder.

The targeting moiety is specific for the donor transplant or subjecttissue to be protected from the immune system. In some embodiments, theeffector molecule binding moiety comprises a de novo generated bindingdomain, e.g. a functional antibody molecule. In some embodiments, theeffector binding/modulating moiety comprises amino acid sequencederiving from the natural ligand that recognizes an inhibitory receptorexpressed on the surface of an immune cell, e.g., a T cell.

In some embodiments, the therapeutic compound silences immune cells,e.g., T cells, proximal to the transplant or donor tissue to beprotected but does not silence immune cells, e.g., T cells, not proximalto the target, as the therapeutic compound requires the presence of thetarget transplant or donor tissue for function. This in contrast to whenthe therapeutic compound binds only to the inhibitory receptor expressedby the immune cell, e.g., T cell, in which case there is no functionalconsequence.

Methods and therapeutic compounds described here are based at least inpart on providing site-specific immune-privilege. Therapeutic compoundsand method of using them described herein allow the minimization, e.g.,the reduction or elimination of, non-site specific systemicadministration of immune-suppressive therapeutic agents in clinicalsettings, e.g., where reversal and suppression of an immune response isdesired, such as in autoimmune diseases or tissue, e.g., organ,transplant. While capable of clinically meaningful response when theunderlying pathophysiology driven by an aberrant immune system isimpacted, broadly acting immunosuppressants have the undesirable effectof reducing the patient's systemic immune system function. As the roleof a normally functioning immune system is to combat the constantbarrage of pathogenic and opportunistic organisms existing in thesurrounding environment and to constantly purge healthy individuals ofcancerous cells, patients undergoing chronic immunosuppression are at anincreased risk to develop infections and cancer. Methods and therapeuticcompounds described herein provide therapies that selectively target andattenuate, reduce, or extinguish only the pathogenic immune response atthe site of pathology while having minimal inhibition of normal systemicimmune system function elsewhere.

In some embodiments, a therapeutic compound is provided as providedherein. In some embodiments, the compound comprises a i) a specifictargeting moiety selected from: a) a donor specific targeting moietywhich, e.g., preferentially binds a donor target; or b) a tissuespecific targeting moiety which, e.g., preferentially binds targettissue of a subject; and ii) an effector binding/modulating moietyselected from: (a) an immune cell inhibitory molecule binding/modulatingmoiety (ICIM binding/modulating moiety); (b) an immunosuppressive immunecell binding/modulating moiety (IIC binding/modulating moiety); or (c)an effector binding/modulating moiety that, as part of a therapeuticcompound, promotes an immuno-suppressive local microenvironment, e.g.,by providing in the proximity of the target, a substance that inhibitsor minimizes attack by the immune system of the target (SMbinding/modulating moiety).

In some embodiments, the effector binding/modulating moiety comprises anICIM binding/modulating moiety. In some embodiments, the effectorbinding/modulating moiety comprises an ICIM binding/modulating moietycomprising an inhibitory immune checkpoint molecule ligand molecule. Insome embodiments, the inhibitory immune molecule counter-ligand moleculecomprises a PD-L1 molecule. In some embodiments, the ICIM is wherein theinhibitory immune molecule counter ligand molecule engages a cognateinhibitory immune checkpoint molecule selected from PD-1, KIR2DL4,LILRB1, LILRB, or CTLA-4. In some embodiments, the ICIM is an antibody.In some embodiments, the ICIM comprises an antibody that binds to PD-1,KIR2DL4, LILRB1, LILRB, or CTLA-4. In some embodiments, the ICIMbinding/modulating moiety which comprises a functional antibody moleculeto a cell surface inhibitory molecule.

In some embodiments, the cell surface inhibitory molecule is aninhibitory immune checkpoint molecule. In some embodiments, theinhibitory immune checkpoint molecule is selected from PD-1, KIR2DL4,LILRB1, LILRB2, CTLA-4, or selected from Table 1.

In some embodiments, the effector binding/modulating moiety comprises anIIC binding/modulating moiety.

In some embodiments, the compound has the formula from N-terminus toC-terminus: R1-Linker Region A-R2 or R3-Linker Region B-R4,

wherein, R1, R2, R3, and R4, each independently comprises an effectorbinding/modulating moiety, e.g., an ICIM binding/modulating moiety, anIIC binding/modulating moiety, ICSM binding/modulating moiety, or an SMbinding/modulating moiety; a specific targeting moiety; or is absent;provided that an effector binding/modulating moiety and a specifictargeting moiety are present.

In some embodiments, polypeptides comprising a targeting moiety thatbinds to a target cell and an effector binding/modulating moiety,wherein the effector binding/modulating moiety is a IL-2 muteinpolypeptide (IL-2 mutein), which is a mutant IL-2 protein, are provided.In some embodiments, the targeting moiety comprises an antibody thatbinds to a target protein on the surface of a target cell. In someembodiments, the polypeptide comprises two polypeptide chains asprovided for herein. In some embodiments, the first chain comprises a VHdomain and the second chain comprises a VL domain of an antibody thatbinds to the target cell or a protein that is expressed on the targetcell, such as, but not limited to, MAdCAM. In some embodiments, thetargeting moiety is an antibody that binds to MAdCAM. In someembodiments, the targeting moiety binds to OAT1 (SLC22A6) and OCT2(SLC22A2). In some embodiments, the targeting moiety is an antibody thatbinds to OAT1 (SLC22A6) and OCT2 (SLC22A2). In some embodiments, thetargeting moiety does not bind to OAT1 (SLC22A6) and OCT2 (SLC22A2). Forthe avoidance of doubt, the OCT2 referenced herein is not thetranscription factor, but rather is the surface protein expressed inkidney tissue. In some embodiments, the targeting moiety is a moietythat specifically binds to a protein found in the pancreas. In someembodiments, the targeting moiety binds to FXYD2, TSPAN7, DPP6,HEPACAM2, TMEM27, or GPR119. In some embodiments, the targetin moietydoes not bind to FXYD2, TSPAN7, DPP6, HEPACAM2, TMEM27, or GPR119. Insome embodiments, the targeting moiety is antibody that binds to FXYD2,TSPAN7, DPP6, HEPACAM2, TMEM27, or GPR119.

In some embodiments, the polypeptide comprises a first chain and asecond chain that form the polypeptide or therapeutic compound, wherein

the first chain comprises:

V_(H)-H_(C)-Linker-C₁, wherein V_(H) is a variable heavy domain thatbinds to the target cell with a V_(L) domain of the second chain; H, isa heavy chain of antibody comprising CH1-CH2-CH3 domain, the Linker is aglycine/serine amino acid sequence as provided herein or is absent, andC₁ is a IL-2 mutein that can be fused to a Fc protein in either theN-terminal or C-terminal orientation as provided for herein, whereinthere can be a glycine/serine linker linking the IL-2 mutein to the Fcprotein; and

the second chain comprises:

V_(L)-L_(c), wherein V_(L) is a variable light chain domain that bindsto the target cell with the V_(H) domain of the first chain, and theL_(c) domain is a light chain CK domain. In some embodiments, the firstchain comprises C₁-Linker-V_(H)-H_(c), with the variables as definedabove.

In some embodiments, the polypeptide comprises the formula ofC₁-linker-CH2-CH3-Linker-scFv, wherein C₁ and the Linker are as definedabove and herein, the CH2 and CH3 are heavy chain domains and the scFvis a single chain antibody like fragment that acts as the targetingmoiety to bind to tissue targets as provided for herein. In someembodiments, the mutein is fused to the Fc region as provided herein andone or more of the linkers are absent. In some embodiments, the Linkeris a glycine/serine linker as provided for herein. In some embodiments,the linker is a peptide sequence.

In some embodiments, methods of treating auto-immune diseases orconditions are provided herein, the methods comprising administering oneor more of the therapeutic compounds or polypeptides provided herein.

In some embodiments, methods of treating diseases or conditionsdescribed herein are provided herein, the methods comprisingadministering one or more of the therapeutic compounds or polypeptidesprovided herein.

In some embodiments, methods of treating a subject with inflammatorybowel disease are provided, the methods comprising administering atherapeutic compound or polypeptides provided herein to the subject totreat the inflammatory bowel disease. In some embodiments, the subjecthas Crohn's disease and or ulcerative colitis.

In some embodiments, methods of treating a subject with auto-immunehepatitis are provided, the methods comprising administering atherapeutic compound or polypeptides as provided herein to the subjectto treat the auto-immune hepatitis.

In some embodiments, methods of treating primary sclerosing cholangitisare provided, the methods comprising administering a therapeuticcompound or polypeptides as provided herein to the subject to treat theprimary sclerosing cholangitis.

In some embodiments, methods of treating (e.g., reducing) inflammationin the intestine are provided, the methods comprising administering atherapeutic compound or polypeptides as provided herein to the subjectto treat the inflammation in the intestine. In some embodiments, theinflammation is in the small intestine. In some embodiments, theinflammation is in the large intestine. In some embodiments, theinflammation is in the bowel or colon.

In some embodiments, methods of treating (e.g., reducing) inflammationin the pancreas are provided, the methods comprising administering atherapeutic compound or polypeptides as provided herein to the subjectto treat the inflammation in the pancreas. In some embodiments, themethods treat pancreatitis.

In some embodiments, methods of treating Type 1 diabetes are provided,the methods comprising administering a therapeutic compound orpolypeptides as provided herein to the subject to treat the Type 1diabetes.

In some embodiments, methods of treating a transplant subject areprovided, the methods comprising administering a therapeuticallyeffective amount of a therapeutic compound or polypeptides as providedherein to the subject, thereby treating a transplant (recipient)subject.

In some embodiments, methods of treating GVHD in a subject having atransplanted a donor tissue are provided, the methods comprisingadministering a therapeutically effective amount of a therapeuticcompound or polypeptides as provided herein to the subject.

In some embodiments, methods of treating a subject having, or at risk,or elevated risk, for having, an autoimmune disorder are provided, themethods comprising administering a therapeutically effective amount of atherapeutic compound or polypeptides as provided herein, therebytreating the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts non-limiting embodiments of the therapeutic compoundsprovided herein.

FIG. 2 depicts a non-limiting illustration of how a therapeutic compoundprovided herein could function.

FIG. 3 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 3A depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 4 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 5 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 6 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 7 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 8 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 9 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 10 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 11 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 12 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 13 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 14 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 15 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 16 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 17 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 18 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

FIG. 19 depicts a non-limiting illustration of the therapeutic compoundsprovided herein.

DETAILED DESCRIPTION

This application incorporates by reference U.S. application Ser. No.15/922,592 filed Mar. 15, 2018 and PCT Application No.PCT/US2018/022675, filed Mar. 15, 2018, each of which is incorporated byreference in its entirety.

As used herein and unless otherwise indicated, the term “about” isintended to mean±5% of the value it modifies. Thus, about 100 means 95to 105.

As used herein and in the appended claims, the singular forms “a”, “an”and “the” include plural reference unless the context clearly dictatesotherwise.

As used herein, the term “about” means that the numerical value isapproximate and small variations would not significantly affect thepractice of the disclosed embodiments. Where a numerical limitation isused, unless indicated otherwise by the context, “about” means thenumerical value can vary by ±10% and remain within the scope of thedisclosed embodiments.

As used herein, the term “animal” includes, but is not limited to,humans and non-human vertebrates such as wild, domestic, and farmanimals.

As used herein, the term “contacting” means bringing together of twoelements in an in vitro system or an in vivo system. For example,“contacting” a therapeutic compound with an individual or patient orcell includes the administration of the compound to an individual orpatient, such as a human, as well as, for example, introducing acompound into a sample containing a cellular or purified preparationcontaining target.

As used herein, the terms “comprising” (and any form of comprising, suchas “comprise”, “comprises”, and “comprised”), “having” (and any form ofhaving, such as “have” and “has”), “including” (and any form ofincluding, such as “includes” and “include”), or “containing” (and anyform of containing, such as “contains” and “contain”), are inclusive oropen-ended and do not exclude additional, unrecited elements or methodsteps. Any composition or method that recites the term “comprising”should also be understood to also describe such compositions asconsisting, consisting of, or consisting essentially of the recitedcomponents or elements.

As used herein, the term “fused” or “linked” when used in reference to aprotein having different domains or heterologous sequences means thatthe protein domains are part of the same peptide chain that areconnected to one another with either peptide bonds or other covalentbonding. The domains or section can be linked or fused directly to oneanother or another domain or peptide sequence can be between the twodomains or sequences and such sequences would still be considered to befused or linked to one another. In some embodiments, the various domainsor proteins provided for herein are linked or fused directly to oneanother or a linker sequences, such as the glycine/serine sequencesdescribed herein link the two domains together.

As used herein, the term “individual,” “subject,” or “patient,” usedinterchangeably, means any animal, including mammals, such as mice,rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses,or primates, such as humans.

As used herein, the term “inhibit” refers to a result, symptom, oractivity being reduced as compared to the activity or result in theabsence of the compound that is inhibiting the result, symptom, oractivity. In some embodiments, the result, symptom, or activity, isinhibited by about, or, at least, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95%, or 99%. An result, symptom, or activity can also beinhibited if it is completely elimination or extinguished.

As used herein, the phrase “in need thereof” means that the subject hasbeen identified as having a need for the particular method or treatment.In some embodiments, the identification can be by any means ofdiagnosis. In any of the methods and treatments described herein, thesubject can be in need thereof. In some embodiments, the subject is inan environment or will be traveling to an environment in which aparticular disease, disorder, or condition is prevalent.

As used herein, the phrase “integer from X to Y” means any integer thatincludes the endpoints. For example, the phrase “integer from X to Y”means 1, 2, 3, 4, or 5.

As used herein, the term “mammal” means a rodent (i.e., a mouse, a rat,or a guinea pig), a monkey, a cat, a dog, a cow, a horse, a pig, or ahuman. In some embodiments, the mammal is a human.

In some embodiments, therapeutic compounds are provided herein. In someembodiments, the therapeutic compound is a protein or a polypeptide,that has multiple chains that interact with one another. Thepolypeptides can interact with one another through non-covalentinteractions or covalent interactions, such as through disulfide bondsor other covalent bonds. Therefore, if an embodiment refers to atherapeutic compound it can also be said to refer to a protein orpolypeptide as provided for herein and vice versa as the contextdictates.

As used herein, the phrase “ophthalmically acceptable” means having nopersistent detrimental effect on the treated eye or the functioningthereof, or on the general health of the subject being treated. However,it will be recognized that transient effects such as minor irritation ora “stinging” sensation are common with topical ophthalmic administrationof drugs and the existence of such transient effects is not inconsistentwith the composition, formulation, or ingredient (e.g., excipient) inquestion being “ophthalmically acceptable” as herein defined. In someembodiments, the pharmaceutical compositions can be ophthalmicallyacceptable or suitable for ophthalmic administration.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen, target, or an epitope means binding that ismeasurably different from a non-specific interaction. Specific bindingcan be measured, for example, by determining binding of a moleculecompared to binding of a control molecule, which generally is a moleculeof similar structure that does not have binding activity. For example,specific binding can be determined by competition with a controlmolecule that is similar to the target.

Specific binding for a particular antigen, target, or an epitope can beexhibited, for example, by an antibody having a K_(D) for an antigen orepitope of at least about 10^(−4M), at least about 10^(−5M), at leastabout 10^(−6 M), at least about 10^(−7M), at least about 10^(−8M), atleast about 10^(−9M), alternatively at least about 10^(−10 M) at leastabout 10^(−11M), at least about 10^(−12M), or greater, where K_(D)refers to a dissociation rate of a particular antibody-targetinteraction. Typically, an antibody that specifically binds an antigenor target will have a K_(D) that is, or at least, 2-, 4-, 5-, 10-, 20-,50-, 100-, 500-, 1000-, 5,000-, 10,000-, or more times greater for acontrol molecule relative to the antigen or epitope.

In some embodiments, specific binding for a particular antigen, target,or an epitope can be exhibited, for example, by an antibody having aK_(A) or K_(a) for a target, antigen, or epitope of at least 2-, 4-, 5-,20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- or more times greater forthe target, antigen, or epitope relative to a control, where K_(A) orK_(a) refers to an association rate of a particular antibody-antigeninteraction.

As provided herein, the therapeutic compounds and compositions can beused in methods of treatment as provided herein. As used herein, theterms “treat,” “treated,” or “treating” mean both therapeutic treatmentand prophylactic measures wherein the object is to slow down (lessen) anundesired physiological condition, disorder or disease, or obtainbeneficial or desired clinical results. For purposes of theseembodiments, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms; diminishment of extent ofcondition, disorder or disease; stabilized (i.e., not worsening) stateof condition, disorder or disease; delay in onset or slowing ofcondition, disorder or disease progression; amelioration of thecondition, disorder or disease state or remission (whether partial ortotal), whether detectable or undetectable; an amelioration of at leastone measurable physical parameter, not necessarily discernible by thepatient; or enhancement or improvement of condition, disorder ordisease. Treatment includes eliciting a clinically significant responsewithout excessive levels of side effects. Treatment also includesprolonging survival as compared to expected survival if not receivingtreatment.

Provided herein are therapeutic compounds, e.g., therapeutic proteinmolecules, e.g., fusion proteins, including a targeting moiety and aneffector binding/modulating moiety, typically as separate domains. Alsoprovided are methods of using and making the therapeutic compounds. Thetargeting moiety serves to localize the therapeutic compound, and thusthe effector binding/modulating moiety, to a site at whichimmune-privilege is desired. The effector binding/modulating moietycomprises one or more of: (a) an immune cell inhibitory moleculebinding/modulating moiety (an ICIM binding/modulating moiety): (b) animmunosuppressive immune cell binding/modulating moiety (an IICbinding/modulating moiety); (c) a soluble molecule binding/modulatingmoiety (a SM binding/modulating moiety) or (d) a molecule that blocks orinhibits immune cell stimulatory molecule binding/modulating moiety(referred to herein as an ICSM binding/modulating moiety). In someembodiments, the ICSM inhibits immune activation by, for example,blocking the interaction between a costimulatory molecule and itscounterstructure. In some embodiments, a therapeutic compound comprises:(a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and(d); or (a), (b), (c), and (d).

The present disclosure provides, for example, molecules that can act asPD-1 agonists. Without being bound to any particular theory, agonism ofPD-1 inhibits T cell activation/signaling and can be accomplished bydifferent mechanisms. For example cross-linking can lead to agonism,bead-bound, functional PD-1 agonists have been described (Akkaya. Ph.D.Thesis: Modulation of the PD-1 pathway by inhibitory antibodysuperagonists. Christ Church College, Oxford, UK, 2012), which is herebyincorporated by reference. Crosslinking of PD-1 with two mAbs that bindnon-overlapping epitopes induces PD-1 signaling (Davis, US2011/0171220), which is hereby incorporated by reference. Anotherexample is illustrated through the use of a goat anti-PD-1 antiserum(e.g. AF1086, R&D Systems) which is hereby incorporated by reference,which acts as an agonist when soluble (Said et al., 2010, Nat Med) whichis hereby incorporated by reference. Non-limiting examples of PD-1agonists that can be used in the present embodiments include, but arenot limited to, UCB clone 19 or clone 10, PD1AB-1, PD1AB-2, PD1AB-3,PD1AB-4 and PD1AB-5, PD1AB-6 (Anaptys/Celgene), PD1-17, PD1-28, PD1-33and PD1-35 (Collins et al, US 2008/0311117 A1 Antibodies against PD-1and uses therefor, which is incorporated by reference), or can be abi-specific, monovalent anti-PD-1/anti-CD3 (Ono), and the like. In someembodiments, the PD-1 agonist antibodies can be antibodies that blockbinding of PD-L1 to PD-1. In some embodiments, the PD-1 agonistantibodies can be antibodies that do not block binding of PD-L1 to PD-1.

PD-1 agonism can be measured by any method, such as the methodsdescribed in the examples. For example, cells can be constructed thatexpress, including stably express, constructs that include a human PD-1polypeptide fused to a b-galactosidase “Enzyme donor” and 2) a SHP-2polypeptide fused to a b-galactosidase “Enzyme acceptor.” Without beingbound by any theory, when PD-1 is engaged, SHP-2 is recruited to PD-1.The enzyme acceptor and enzyme donor form a fully active b-galactosidaseenzyme that can be assayed. Although, the assay does not directly showPD-1 agonism, but shows activation of PD-1 signaling. PD-1 agonism canalso be measured by measuring inhibition of T cell activation because,without being bound to any theory, PD-1 agonism inhibitsanti-CD3-induced T cell activation. For example, PD-1 agonism can bemeasured by preactivating T cells with PHA (for human T cells) or ConA(for mouse T cells) so that they express PD-1. The cells can then bereactivated with anti-CD3 in the presence of anti-PD-1 (or PD-L1) forthe PD-1 agonism assay. T cells that receive a PD-1 agonist signal inthe presence of anti-CD3 will show decreased activation, relative toanti-CD3 stimulation alone. Activation can be readout by proliferationor cytokine production (IL-2, IFNg, IL-17) or other markers, such asCD69 activation marker. Thus, PD-1 agonism can be measured by eithercytokine production or cell proliferation. Other methods can also beused to measure PD-1 agonism.

PD-1 is Ig superfamily member expressed on activated T cells and otherimmune cells. The natural ligands for PD-1 appear to be PD-L1 and PD-L2.Without being bound to any particular theory, when PD-L1 or PD-L2 bindto PD-1 on an activated T cell, an inhibitory signaling cascade isinitiated, resulting in attenuation of the activated T effector cellfunction. Thus, blocking the interaction between PD-1 on a T cell, andPD-L1/2 on another cell (eg tumor cell) with a PD-1 antagonist is knownas checkpoint inhibition, and releases the T cells from inhibition. Incontrast, PD-1 agonist antibodies can bind to PD-1 and send aninhibitory signal and attenuate the function of a T cell. Thus, PD-1agonist antibodies can be incorporated into various embodimentsdescribed herein as an effector molecule binding/modulating moiety,which can accomplish localized tissue-specific immunomodulation whenpaired with a targeting moiety.

The effector molecule binding/modulating moiety can provide animmunosuppressive signal or environment in a variety of ways. In someembodiments, the effector binding/modulating moiety comprises an ICIMbinding/modulating moiety that directly binds and (under the appropriateconditions as described herein) activates an inhibitory receptorexpressed by immune cells responsible for driving disease pathology. Inanother embodiment the effector binding/modulating moiety comprises andIIC binding/modulating moiety and binds and accumulatesimmunosuppressive immune cells. In some embodiments, the accumulatedimmune suppressive cells promote immune privilege. In another embodimentthe effector binding/modulating moiety comprises an SMbinding/modulating moiety which manipulates the surroundingmicroenvironment to make it less permissible for the function of immunecells, e.g., immune cells driving disease pathology. In someembodiments, the SM binding/modulating moiety depletes an entity thatpromotes immune attack or activation. In some embodiments the effectorbinding/modulating moiety comprises an ICSM binding/modulating moietythat binds a member of a pair of stimulatory molecules, e.g.,costimulatory molecules, and inhibits the interaction between thecostimulatory molecule and the costimulatory molecule counterstructure,such as, but not limited to, OX40 or CD30 or CD40 and OX40L, or CD30L orCD40L and inhibits the immune stimulation of a cell, such as, but notlimited to, a T cell, B cell, NK cell, or other immune cell comprising amember of the pair.

The targeting moiety and effector binding/modulating moiety arephysically tethered, covalently or non-covalently, directly or through alinker entity, to one another, e.g., as a member of the same proteinmolecule in a therapeutic protein molecule. In some embodiments, thetargeting and effector moieties are provided in a therapeutic proteinmolecule, e.g., a fusion protein, typically as separate domains. In someembodiments, the targeting moiety, the effector binding/modulatingmoiety, or both each comprises a single domain antibody molecule, e.g.,a camelid antibody VHH molecule or human soluble VH domain. It may alsocontain a single-chain fragment variable (scFv) or a Fab domain. In someembodiments, the therapeutic protein molecule, or a nucleic acid, e.g.,an mRNA or DNA, encoding the therapeutic protein molecule, can beadministered to a subject. In some embodiments, the targeting andeffector molecule binding/modulating moieties are linked to a thirdentity, e.g., a carrier, e.g., a polymeric carrier, a dendrimer, or aparticle, e.g., a nanoparticle. The therapeutic compounds can be used todown regulate an immune response at or in a tissue at a selected targetor site while having no or substantially less immunosuppressive functionsystemically. The target or site can comprise donor tissue or autologoustissue.

Provided herein are methods of providing site-specific immune privilegefor a transplanted donor tissue, e.g., an allograft tissue, e.g., atissue described herein, e.g., an allograft liver, an allograft kidney,an allograft heart, an allograft pancreas, an allograft thymus or thymictissue, allograft skin, or an allograft lung, with therapeutic compoundsdisclosed herein. In embodiments the treatment minimizes rejection of,minimizes immune effector cell mediated damage to, prolongs acceptanceof, or prolongs the functional life of, donor transplant tissue.

Also provided herein are methods of inhibiting graft versus host disease(GVHD) by minimizing the ability of donor immune cells, e.g., donor Tcells, to mediate immune attack of recipient tissue, with therapeuticcompounds disclosed herein.

Also provided herein are methods of treating, e.g., therapeuticallytreating or prophylactically treating (or preventing), an auto-immunedisorder or response in a subject by administration of a therapeuticcompound disclosed herein, e.g., to provide site or tissue specificmodulation of the immune system. In some embodiments, the methodprovides tolerance to, minimization of the rejection of, minimization ofimmune effector cell mediated damage to, or prolonging a function of,subject tissue. In some embodiments, the therapeutic compound includes atargeting moiety that targets, e.g., specifically targets, the tissueunder, or at risk for, autoimmune attack. Non-limiting exemplary tissuesinclude, but are not limited to, the pancreas, myelin, salivary glands,synoviocytes, and myocytes.

As used herein, the terms “treat,” “treated,” or “treating” in regardsto therapeutic treatment wherein the object is to slow down (lessen) anundesired physiological condition, disorder or disease, or obtainbeneficial or desired clinical results. For example, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms; diminishment of extent of condition, disorder or disease;stabilized (i.e., not worsening) state of condition, disorder ordisease; delay in onset or slowing of condition, disorder or diseaseprogression; amelioration of the condition, disorder or disease state orremission (whether partial or total), whether detectable orundetectable; an amelioration of at least one measurable physicalparameter, not necessarily discernible by the patient; or enhancement orimprovement of condition, disorder or disease. Treatment includeseliciting a clinically significant response without excessive levels ofside effects. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment. Thus, “treatment of anauto-immune disease/disorder” means an activity that alleviates orameliorates any of the primary phenomena or secondary symptomsassociated with the auto-immune disease/disorder or other conditiondescribed herein. The various disease or conditions are provided herein.The therapeutic treatment can also be administered prophylactically topreventing or reduce the disease or condition before the onset.

In some embodiments, administration of the therapeutic compound beginsafter the disorder is apparent. In some embodiments, administration ofthe therapeutic compound, begins prior to onset, or full onset, of thedisorder. In some embodiments, administration of the therapeuticcompound, begins prior to onset, or full onset, of the disorder, e.g.,in a subject having the disorder, a high-risk subject, a subject havinga biomarker for risk or presence of the disorder, a subject having afamily history of the disorder, or other indicator of risk of, orasymptomatic presence of, the disorder. For example, In someembodiments, a subject having islet cell damage but which is not yetdiabetic, is treated.

While not wishing to be bound by theory, it is believed that thetargeting moiety functions to bind and accumulate the therapeutic to atarget selectively expressed at the anatomical site where immuneprivilege is desired. In some embodiments, e.g., in the context of donortissue transplantation, the target moiety binds to a target, e.g., anallelic product, present in the donor tissue but not the recipient. Fortreatment of autoimmune disorders, the targeting moiety binds a targetpreferentially expressed at the anatomical site where immune privilegeis desired, e.g., in the pancreas. For treatment of GVHD, the targetingmoiety targets the host tissue, and protects the host against attackfrom transplanted immune effector cells derived from transplantedtissue.

Again, while not wishing to be bound by theory it is believed that theeffector binding/modulating moiety serves to deliver animmunosuppressive signal or otherwise create an immune privilegedenvironment.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which these embodiments belong. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present embodiments, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.Headings, sub-headings or numbered or lettered elements, e.g., (a), (b),(i) etc, are presented merely for ease of reading. The use of headingsor numbered or lettered elements in this document does not require thesteps or elements be performed in alphabetical order or that the stepsor elements are necessarily discrete from one another. Other features,objects, and advantages of the embodiments will be apparent from thedescription and drawings, and from the claims.

Additional Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the embodiments pertains. In describing and claimingthe present embodiments, the following terminology and terminologyotherwise referenced throughout the present application will be usedaccording to how it is defined, where a definition is provided.

It is also to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting.

Antibody molecule, as that term is used herein, refers to a polypeptide,e.g., an immunoglobulin chain or fragment thereof, comprising at leastone functional immunoglobulin variable domain sequence. An antibodymolecule encompasses antibodies (e.g., full-length antibodies) andantibody fragments. In some embodiments, an antibody molecule comprisesan antigen binding or functional fragment of a full length antibody, ora full length immunoglobulin chain. For example, a full-length antibodyis an immunoglobulin (Ig) molecule (e.g., an IgG antibody) that isnaturally occurring or formed by normal immunoglobulin gene fragmentrecombinatorial processes). In embodiments, an antibody molecule refersto an immunologically active, antigen-binding portion of animmunoglobulin molecule, such as an antibody fragment. An antibodyfragment, e.g., functional fragment, comprises a portion of an antibody,e.g., Fab, Fab′, F(ab′)₂, F(ab)2, variable fragment (Fv), domainantibody (dAb), or single chain variable fragment (scFv). A functionalantibody fragment binds to the same antigen as that recognized by theintact (e.g., full-length) antibody. The terms “antibody fragment” or“functional fragment” also include isolated fragments consisting of thevariable regions, such as the “Fv” fragments consisting of the variableregions of the heavy and light chains or recombinant single chainpolypeptide molecules in which light and heavy variable regions areconnected by a peptide linker (“scFv proteins”). In some embodiments, anantibody fragment does not include portions of antibodies withoutantigen binding activity, such as Fc fragments or single amino acidresidues. Exemplary antibody molecules include full length antibodiesand antibody fragments, e.g., dAb (domain antibody), single chain, Fab,Fab′, and F(ab′)2 fragments, and single chain variable fragments(scFvs).

The term “antibody molecule” also encompasses whole or antigen bindingfragments of domain, or single domain, antibodies, which can also bereferred to as “sdAb” or “VHH.” Domain antibodies comprise either V_(H)or V_(L) that can act as stand-alone, antibody fragments. Additionally,domain antibodies include heavy-chain-only antibodies (HCAbs). Domainantibodies also include a CH2 domain of an IgG as the base scaffold intowhich CDR loops are grafted. It can also be generally defined as apolypeptide or protein comprising an amino acid sequence that iscomprised of four framework regions interrupted by three complementaritydetermining regions. This is represented asFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. sdAbs can be produced in camelids suchas llamas, but can also be synthetically generated using techniques thatare well known in the art. The numbering of the amino acid residues of asdAb or polypeptide is according to the general numbering for VH domainsgiven by Kabat et al. (“Sequence of proteins of immunological interest,”US Public Health Services, NIH Bethesda, Md., Publication No. 91, whichis hereby incorporated by reference). According to this numbering, FR1of a sdAb comprises the amino acid residues at positions 1-30, CDR1 of asdAb comprises the amino acid residues at positions 31-36, FR2 of a sdAbcomprises the amino acids at positions 36-49, CDR2 of a sdAb comprisesthe amino acid residues at positions 50-65, FR3 of a sdAb comprises theamino acid residues at positions 66-94, CDR3 of a sdAb comprises theamino acid residues at positions 95-102, and FR4 of a sdAb comprises theamino acid residues at positions 103-113. Domain antibodies are alsodescribed in WO2004041862 and WO2016065323, each of which is herebyincorporated by reference. The domain antibodies can be a targetingmoiety as described herein.

Antibody molecules can be monospecific (e.g., monovalent or bivalent),bispecific (e.g., bivalent, trivalent, tetravalent, pentavalent, orhexavalent), trispecific (e.g., trivalent, tetravalent, pentavalent,hexavalent), or with higher orders of specificity (e.g, tetraspecific)and/or higher orders of valency beyond hexavalency. An antibody moleculecan comprise a functional fragment of a light chain variable region anda functional fragment of a heavy chain variable region, or heavy andlight chains may be fused together into a single polypeptide.

Examples of formats for multispecific therapeutic compounds, e.g.,bispecific antibody molecules are shown in the following non-limitingexamples. Although illustrated with antibody molecules, they can be usedas platforms for therapeutic molecules that include other non-antibodymoieties as specific binding or effector moieties. In some embodiments,these non-limiting examples are based upon either a symmetrical orasymmetrical Fc formats.

For example, the figures illustrate non-limiting and varied symmetrichomodimer approach. In some embodiments, the dimerization interfacecenters around human IgG1 CH2-CH3 domains, which dimerize via a contactinterface spanning both CH2/CH2 and CH3/CH3. The resulting bispecificantibodies shown have a total valence comprised of four binding unitswith two identical binding units at the N-terminus on each side of thedimer and two identical units at the C-terminus on each side of thedimer. In each case the binding units at the N-terminus of thehomo-dimer are different from those at the C-terminus of the homo-dimer.Using this type of bivalency for both an inhibitory T cell receptor ateither terminus of the molecule and bivalency for a tissue tetheringantigen can be achieved at either end of the molecule.

For example, in FIG. 3, a non-limiting embodiment is illustrated. TheN-terminus of the homodimer contains two identical Fab domains comprisedof two identical light chains, which are separate polypeptides,interfaced with the n-terminal VH-CH1 domains of each heavy chain viathe VH/VL interaction and Ckappa or Clambda interaction with CH1. Thenative disulphide bond between the Ckappa or Clambda with CH1 is presentproviding a covalent anchor between the light and heavy chains. At thec-terminus of this design are two identical scFv units where by (in thisexample) the c-terminus of the CH3 domain of the Fc, is followed by aflexible, hydrophilic linker typically comprised of (but not limited to)serine, glycine, alanine, and/or threonine residues, which is followedby the VH domain of each scFv unit, which is followed by aglycine/serine rich linker, followed by a VL domain. These tandem VH andVL domains associate to form a single chain fragment variable (scFv)appended at the c-terminus of the Fc. Two such units exist at thec-terminus of this molecule owing to the homodimeric nature centered atthe Fc. The domain order of scFvs may be configured to be from N to Cterminus either VH-Linker-VL or VL-Linker-VH.

A non-limiting example of a molecule that has different binding regionson the different ends is where, one end is a PD-1 agonist and theantibody that provides target specificity is an anti-MAdCAM-1 antibody.This can be illustrated as shown, for example, in FIG. 3A, whichillustrates the molecules in different orientations.

In some embodiments, the MAdCAM antibody is a blocking or non-blockingantibody as described elsewhere herein. Without being bound to anytheory, MAdCAM has been shown to interact with the headpiece of theintegrin α4β7 expressed on lymphocytes via multiple residues within itstwo Ig superfamily I-set domains and the atomic level structural basisfor that interaction has been described (Viney J L et al. (1996). JImmunol. 157, 2488-2497; Yu Y et al (2013). J Biol Chem. 288, 6284-6294;Yu Y et al (2012). J Cell Biol. 196, 131-146, each of which isincorporated by reference in its entirety). It has been shown in greatstructural, mechanistic and functional detail in both the human (Chen Jet al (2003). Nat Struct Biol. 10, 995-1001; de Chateau M et al (2001).Biochemistry. 40, 13972-13979) and mouse (Day E S et al (2002). CellCommun Adhes. 9, 205-219; Hoshino H et al (2011). J Histochem Cytochem.59, 572-583) molecular systems that any interaction of MAdCAM with α4β7is dependent on three dication binding sites present in the integrinbeta 7 sub unit I-like domain and that these metal binding sites cancoordinate with Ca2+, Mn2+, and Mg2+. Using cell adhesion assays, flowcytometry, and/or flow chamber assays in the presence of high levels ofCa2+ with or without Mg2+ or Mn2+, the MAdCAM/α4β7 interaction is shownto be of a lower functional affinity and permits rolling adhesion oflymphocytes, whereas in low Ca2+ but higher Mg2+ or Mn2+ which activatesthe integrin, the MAdCAM/α4β7 interaction is of a higher functionalaffinity and mediates firm lymphocyte adhesion (Chen J et al (2003). NatStruct Biol. 10, 995-1001). A number of groups have shown that variouscell:cell, cell:membrane prep, and/or cell:protein basedadhesion/interaction assays can be utilized, with FACS, cell flowchamber based counts, or IHC based read-outs to monitor the impact ofanti-MAdCAM or anti-α4β7 antibodies upon the interaction of MAdCAM withα4β7, allowing one to identify blocking or non-blocking antibodies(Nakache, M et al (1989). Nature. 337, 179-181; Streeter, P R et al(1988). Nature. 331. 41-46; Yang Y et al (1995). Scand J Immunol. 42.235-247; Leung E et al (2004). Immunol Cell Biol. 82. 400-409; Pullen Net al (2009). B J Pharmacol. 157. 281-293; Soler D et al (2009). JPharmacol Exp Ther. 330. 864-875; Qi J et al (2012). J Biol Chem. 287.15749-15759).

This has been exemplified in the mouse system setting with theidentification of anti-mouse MAdCAM antibodies such as MECA-89(non-blocking) and MECA-367 (blocking)) Nakache, M et al (1989). Nature.337, 179-181; Streeter, P R et al (1988). Nature. 331. 41-46; Yang Y etal (1995). Scand J Immunol. 42. 235-247). In a human system, antibodieshave been identified that block the interaction of human MAdCAM withhuman α4β7 such as anti-human MAdCAM PF-00547659 (Pullen N et al (2009).B J Pharmacol. 157. 281-293) and anti-human α4β7 vedolizumab (Soler D etal (2009). J Pharmacol Exp Ther. 330. 864-875), as well as antibodiesthat do not block the interaction such as anti-human MAdCAM clone 17F5(Soler D et al (2009). J Pharmacol Exp Ther. 330. 864-875), andanti-human α4β7 clone J19 (Qi J et al (2012). J Biol Chem. 287.15749-15759). Thus, the antibody can either be blocking or non-blockingbased upon the desired effect. In some embodiments, the antibody is anon-blocking MAdCAM antibody. In some embodiments, the antibody is ablocking MAdCAM antibody. One non-limiting example of demonstratingwhether an antibody is blocking or non-blocking can be found in Example6, but any method can be used. Each of the references described hereinare incorporated by reference in its entirety. In some embodiments, thePD-1 Agonist is replaced with an IL-2 mutein, such as, but not limitedto, the ones described herein.

In another example, and as depicted in FIG. 4, the N-terminus of thehomodimer contains two identical Fab domains comprised of two identicallight chains, which are separate polypeptides, interfaced with then-terminal VH-CH1 domains of each heavy chain via the VH/VL interactionand Ckappa or Clambda interaction with CH1. The native disulphide bondbetween the Ckappa or Clambda with CH1 is present providing a covalentanchor between the light and heavy chains. At the c-terminus of thisdesign are two identical VH units (though non-antibody moieties couldalso be substituted here or at any of the four terminalattachment/fusion points) where by (in this example) the c-terminus ofthe CH3 domain of the Fc, is followed by a flexible, hydrophilic linkertypically comprised of (but not limited to) serine, glycine, alanine,and/or threonine residues, which is followed by a soluble independentVH3 germline family based VH domain. Two such units exist at thec-terminus of this molecule owing to the homodimeric nature centered atthe Fc.

In another non-limiting example, as depicted in FIG. 5, the N-terminusof the homodimer contains two identical Fab domains comprised of twoidentical light chains, which, unlike FIG. 3 and FIG. 4, are physicallyconjoined with the heavy chain at the N-terminus via a linker betweenthe c-terminus of Ckappa or Clambda and the N-terminus of the VH. Thelinker may be 36-80 amino acids in length and comprised of serine,glycine, alanine and threonine residues. The physically conjoinedn-terminal light chains interface with the n-terminal VH-CH1 domains ofeach heavy chain via the VH/VL interaction and Ckappa or Clambdainteraction with CH1. The native disulphide bond between the Ckappa orClambda with CH1 is present providing additional stability between thelight and heavy chains. At the c-terminus of this design are twoidentical Fab units where by (in this example) the c-terminus of the CH3domain of the Fc, is followed by a flexible, hydrophilic linkertypically comprised of (but not limited to) serine, glycine, alanine,and/or threonine residues, which is followed by a CH1 domain, followedby a VH domain at the c-terminus. The light chain that is designed topair with the c-terminal CH1/VH domains is expressed as a separatepolypeptide, unlike the N-terminal light chain which is conjoined to then-terminal VH/CH1 domains as described. The C-terminal light chains forman interface at between VH/VL and Ckappa or Clambda with CH1. The nativedisulphide anchors this light chain to the heavy chain. Again, any ofthe antibody moieties at any of the four attachment/fusion points can besubstituted with a non-antibody moiety, e.g., a effectorbinding/modulating moiety that does not comprise an antibody molecule.

The bispecific antibodies can also be asymmetric as shown in thefollowing non-limiting examples. Non-limiting example are also depictedin FIG. 6, FIG. 7, and FIG. 8, which illustrate anasymmetric/heterodimer approach. Again, in any of these formats, any ofthe antibody moieties at any of the four attachment/fusion points can besubstituted with a non-antibody moiety, e.g., a effectorbinding/modulating moiety that does not comprise an antibody molecule.In some embodiments, the dimerization interface centers around the humanIgG1 CH2-CH3 domains, which dimerize via a contact interface spanningboth CH2/CH2 and CH3/CH3. However, in order to achieveheterodimerization instead of homodimerization of each heavy chain,mutations are introduced in each CH3 domain. The heterodimerizingmutations include T366W mutation (kabat) in one CH3 domain and T366S,L368A, and Y407V (kabat) mutations in the other CH3 domain. Theheterodimerizing interface may be further stabilized with de novodisulphide bonds via mutation of native residues to cysteine residuessuch as 5354 and Y349 on opposite sides of the CH3/CH3 interface. Theresulting bispecific antibodies shown have a total valence comprised offour binding units. With this approach, the overall molecule can bedesigned to have bispecificity at just one terminus and monospecificityat the other terminus (trispecificity overall) or bispecificity ateither terminus with an overall molecular specificity of 2 or 4. In theillustrative examples below, the C-terminus comprises two identicalbinding domains which could, for example, provide bivalentmonospecificity for a tissue tethering target. At the N-terminus of allthree of the illustrative examples, both binding domains comprisedifferent recognition elements/paratopes and which could achieverecognition of two different epitopes on the same effector moietytarget, or could recognize for examples a T cell inhibitory receptor andCD3. In some embodiments, the N-terminal binding moieties may beinterchanged with other single polypeptide formats such as scFv, singlechain Fab, tandem scFv, VH or VHH domain antibody configurations forexample. Other types of recognition element may be used also, such aslinear or cyclic peptides.

An example of an asymmetric molecule is depicted in FIG. 6. Referring toFIG. 6, the N-terminus of the molecule is comprised of a first lightchain paired with a first heavy chain via VH/VL and Ckappa orClambda/CH1 interactions and a covalent tether comprised of the nativeheavy/light chain disulphide bond. On the opposite side of thisheterodimeric molecule at the N-terminus is a second light chain and asecond heavy chain which are physically conjoined via a linker betweenthe c-terminus of Ckappa or Clambda and the N-terminus of the VH. Thelinker may be 36-80 amino acids in length and comprised of serine,glycine, alanine and threonine residues. The physically conjoinedn-terminal light chains interface with the n-terminal VH-CH1 domains ofeach heavy chain via the VH/VL interaction and Ckappa or Clambdainteraction with CH1. The native disulphide bond between the Ckappa orClambda with CH1 is present providing additional stability between thelight and heavy chains. At the c-terminus of the molecule are twoidentical soluble VH3 germline family VH domains joined via anN-terminal glycine/serine/alanine/threonine based linker to thec-terminus of the CH3 domain of both heavy chain 1 and heavy chain 2.

In some embodiments, an asymmetric molecule can be as illustrated asdepicted in FIG. 7. For example, the N-terminus of the molecule iscomprised of two different VH3 germlined based soluble VH domains linkedto the human IgG1 hinge region via a glycine/serine/alanine/threoninebased linker. The VH domain connected to the first heavy chain isdifferent to the VH domain connected to the second heavy chain. At thec-terminus of each heavy chain is an additional soluble VH3 germlinebased VH domain, which is identical on each of the two heavy chains. Theheavy chain heterodimerizes via the previously described knobs intoholes mutations present at the CH3 interface of the Fc module.

In some embodiments, an asymmetric molecule can be as illustrated inFIG. 8. This example is similar to the molecule shown in FIG. 7, exceptboth N-terminal Fab units are configured in a way that light chain 1 andlight chain 2 are physically conjoined with heavy chain 1 and heavychain 2 via a linker between the c-terminus of Ckappa or Clambda and theN-terminus of each respective VH. The linker in each case may be 36-80amino acids in length and comprised of serine, glycine, alanine andthreonine residues. The physically conjoined n-terminal light chainsinterface with the n-terminal VH-CH1 domains of each heavy chain via theVH/VL interaction and Ckappa or Clambda interaction with CH1. The nativedisulphide bond between the Ckappa or Clambda with CH1 is presentproviding additional stability between the light and heavy chains.

Bi-specific molecules can also have a mixed format. This is illustrated,for example, in FIG. 9, FIG. 10, and FIG. 11.

For example, as illustrated in FIG. 9, illustrates a homodimer Fc basedapproach (see FIGS. 3, 4, and 5), combined with the moiety formatselection of FIG. 7, whereby the total molecular valency is four, butspecificity is restricted to two specificities. The N-terminus iscomprised of two identical soluble VH3 germline based VH domains and thec-terminus is comprised of two identical soluble VH3 germlined based VHdomains of different specificity to the N-terminal domains. Therefore,each specificity has a valence of two. Again, in this format, any of theantibody moieties at any of the four attachment/fusion points can besubstituted with a non-antibody moiety, e.g., an effectorbinding/modulating moiety that does not comprise an antibody molecule.

FIG. 10 illustrates another example. In this example, the molecule iscomprised of four VH3 germline based soluble VH domains. The first twodomains have the same specificity (for example an inhibitory receptor),the 3rd domain from the N-terminus may have specificity for a tissueantigen and the fourth domain from the N-terminus may have specificityfor human serum albumin (HSA), thereby granting the molecule extendedhalf-life in the absence of an Ig Fc domain. Three glycine, serine,alanine and/or threonine rich linkers exists between domains 1 and 2,domains 2 and 3, and domains 3 and 4. This format may be configured withup to tetraspecificity, but monovalent in each case, or to havebispecificity with bivalency in each case. The order of domains can bechanged. Again, in this format, any of the antibody moieties can besubstituted with a non-antibody moiety, e.g., a effectorbinding/modulating moiety that does not comprise an antibody molecule.

FIG. 11 illustrates yet another approach. This example is similar toFIGS. 3 and 4, in that it is Fc homodimer based with two identical Fabunits (bivalent monospecificity) at the N-terminus of the molecule. Thisexample differs in that the C-terminus of each heavy chain is appendedwith a tandem-scFv. Thus, in each case the c-terminus of the CH3 domainof the Fc is linked via a glycine/serine/alanine/threonine based linkerto the N-terminus of a first VH domain, which is linked via theC-terminus by a 12-15 amino acid glycine/serine rich linker to theN-terminus of a first VL domain, which linked via a 25-35 amino acidglycine/serine/alanine/threonine based linker at the c-terminus to theN-terminus of a second VH domain, which is linked via the c-terminuswith a 12-15 amino acid glycine/serine based linker to the N-terminus ofa 2nd VL domain. In this Fc homodimer based molecule there are thereforetwo identical tandem scFvs at the c-terminus of the molecule offeringeither tetravalency for a single tissue antigen for example or bivalencyto two different molecules. This format could also be adapted with aheterodimer Fc core allowing two different tandem-scFvs at thec-terminus of the Fc allowing for monovalent tetraspecificity at thec-terminus while retaining either bivalent monospecificity at theN-terminus or monovalent bispecificity at the N-terminal via usage ofsingle chain Fab configurations as in FIGS. 5, 6, and 7. This moleculecan therefore be configured to have 2, 3, 4, 5, or 6 specificities. Thedomain order of scFvs within the tandem scFv units may be configured tobe from N to C terminus either VH-Linker-VL or VL-Linker-VH. Again, inthis format, any of the antibody moieties at any of the fourattachment/fusion points can be substituted with a non-antibody moiety,e.g., a effector binding/modulating moiety that does not comprise anantibody molecule.

Bi-specific antibodies can also be constructed to have, for example,shorter systemic PK while having increased tissue penetration. Thesetypes of antibodies can be based upon, for example, a human VH3 baseddomain antibody format. These are illustrated, for example, in FIGS. 12,13, and 14. FIGS. 12, 13, and 14 each comprised a soluble VH3 germlinefamily based VH domain modules. Each domain is approximately 12.5 kDaallowing for a small overall MW, which, without being bound to anyparticular theory, should be beneficial for enhanced tissue penetration.In these examples, none of the VH domains recognize any half-lifeextending targets such as FcRn or HSA. As illustrated in FIG. 12, themolecule is comprised of two VH domains joined with a flexiblehydrophilic glycine/serine based linker between the C-terminus of thefirst domain and N-terminus of the second domain. In this example onedomain may recognize a T cell co-stimulatory receptor and the second mayrecognize a tissue tethering antigen. As illustrated in FIG. 13, themolecule is comprised of three VH domains with N-C terminal linkages ofhydrophilic glycine/serine based linkers. The molecule may be configuredto be trispecific but monovalent for each target. It may be bispecificwith bivalency for one target and monovalency for another. Asillustrated in FIG. 14, the molecule is comprised of four VH domainswith N-C terminal Glycine/Serine rich linkers between each domain. Thismolecule may be configured to be tetraspecific, trispecific, orbispecific with varying antigenic valencies in each case. Again, in thisformat, any of the antibody moieties at can be substituted with anon-antibody moiety, e.g., a effector binding/modulating moiety thatdoes not comprise an antibody molecule.

Other embodiments of bi-specific antibodies are illustrated in FIGS. 15and 16. FIGS. 15 and 16 are comprised of the naturally heterodimerizingcore of the human IgG CH1/Ckappa interface, including the c-terminalheavy/light disulphide bond which covalently anchors the interaction.This format does not contain an Fc or any moieties for half lifeextension. As illustrated in FIG. 15, the molecule, at the N-terminus ofthe constant kappa domain is appended with an scFv fragment consistingof an N-terminal VH domain, linked at its C-terminus to the N-terminusof a VL domain via a 12-15 amino acid gly/ser based linker, which islinked by its C-terminus to the N-terminus of the constant kappa domainvia the native VL-Ckappa elbow sequence. The CH1 domain is appended atthe N-terminus with an scFv fragment consisting of an N-terminal VLdomain linked at its c-terminus via a 12-15 amino acid gly/ser linker tothe N-terminus of a VH domain, which is linked at its c-terminus to theN-terminus of the CH1 domains via the natural VH-CH1 elbow sequence. Asillustrated in FIG. 16, the molecule has the same N-terminalconfiguration to Example 13. However the C-terminus of the constantkappa and CH1 domains are appended with scFv modules which may be ineither the VH-VL or VL-VH configuration and may be either specific forthe same antigen or specific for two different antigens. The VH/VLinter-domain linkers may be 12-15 amino acids in length and consistingof gly/ser residues. The scFv binding sub-units may be swapped forsoluble VH domains, or peptide recognition elements, or even tandem-scFvelements. This approach can also be configured to use variable lambdaand/or constant lambda domains. Again, in this format, any of theantibody moieties at any of the attachment/fusion points can besubstituted with a non-antibody moiety, e.g., a effectorbinding/modulating moiety that does not comprise an antibody molecule.

FIG. 17 illustrates another embodiment. FIG. 17 represents a tandem scFvformat consisting of a first N-terminal VL domain linked at itsC-terminus to the N-terminus of a first VH domain with a 12-15 aminoacid gly/ser rich linker, followed at the first VH c-terminus by a 25-30amino acid gly/ser/ala/thr based linker to the N-terminus of a second VLdomain. The second VL domain is linked at the C-terminus to theN-terminus of a 2nd VH domain by a 12-15 amino acid gly/ser linker. EachscFv recognizes a different target antigen such as a co-stimulatory Tcell molecule and a tissue tethering target. Again, in this format, anyof the antibody moieties can be substituted with a non-antibody moiety,e.g., a effector binding/modulating moiety that does not comprise anantibody molecule.

FIG. 18 illustrates another embodiment. FIG. 18 is a F(ab′)2 scFvfusion. This consists of two identical Fab components joined via twodisulphide bonds in the native human IgG1 hinge region c-terminal of thehuman IgG CH1 domain. The human IgG1 CH2 and CH3 domains are absent. Atthe c-terminus of heavy chains 1 and 2 are two identical scFv fragmentslinked via a gly/ser/ala/thr rich linker to the c-terminus of the huIgG1hinge region. In the configuration shown, the VH is N-terminal in eachscFv unit and linked via a 12-15 amino acid gly/ser rich linker to theN-terminus of a VL domain. An alternative configuration would beN-term-VL-Linker-VH-C-term. In this design, the construct is bispecificwith bivalency for reach target. Again, in this format, any of theantibody moieties at any of the four attachment/fusion points can besubstituted with a non-antibody moiety, e.g., a effectorbinding/modulating moiety that does not comprise an antibody molecule.

CD39 molecule, as that term as used herein, refers to a polypeptidehaving sufficient CD39 sequence that, as part of a therapeutic compound,it phosphohydrolyzes ATP to AMP. In some embodiments, a CD39 moleculephosphohydrolizes ATP to AMP equivalent to, or at least, 10, 20, 30, 40,50, 60, 70, 80, 90, or 95% of the rate of a naturally occurring CD39,e.g., the CD39 from which the CD39 molecule was derived. In someembodiments, a CD39 molecule has at least 60, 70, 80, 90, 95, 99, or100% sequence identity, or substantial sequence identity, with anaturally occurring CD39.

Any functional isoform can be used (with CD39 or other proteinsdiscussed herein). Exemplary CD39 sequence include Genbank accession#NP_001767.3 or a mature form from the following sequence:

(SEQ ID NO: 1) MEDTKESNVKTFCSKNILAILGFSSIIAVIALLAVGLTQNKALPENVKYGIVLDAGSSHTSLYIYKWPAEKENDTGVVHQVEECRVKGPGISKFVQKVNEIGIYLTDCMERAREVIPRSQHQETPVYLGATAGMRLLRMESEELADRVLDVVERSLSNYPFDFQGARIITGQEEGAYGWITINYLLGKFSQKTRWFSIVPYETNNQETFGALDLGGASTQVTFVPQNQTIESPDNALQFRLYGKDYNVYTHSFLCYGKDQALWQKLAKDIQVASNEILRDPCFHPGYKKVVNVSDLYKTPCTKRFEMTLPFQQFEIQGIGNYQQCHQSILELFNTSYCPYSQCAFNGIFLPPLQGDFGAFSAFYFVMKFLNLTSEKVSQEKVTEMMKKFCAQPWEEIKTSYAGVKEKYLSEYCFSGTYILSLLLQGYHFTADSWEHIHFIGKIQGSDAGWTLGYMLNLTNMIPAEQPLSTPLSHSTYVFLMVLFSLVLFTVAIIGLLIFH KPSYFWKDMV.

In some embodiments, a CD39 molecule comprises a soluble catalyticallyactive form of CD39 found to circulate in human or murine serum, see,e.g., Metabolism of circulating ADP in the bloodstream is mediated viaintegrated actions of soluble adenylate kinase-1 and NTPDase1/CD39activities, Yegutkin et al. FASEB J. 2012 September; 26(9):3875-83. Asoluble recombinant CD39 fragment is also described in Inhibition ofplatelet function by recombinant soluble ecto-ADPase/CD39, Gayle, etal., J Clin Invest. 1998 May 1; 101(9): 1851-1859.

CD73 molecule, as that term as used herein, refers to a polypeptidehaving sufficient CD73 sequence that, as part of a therapeutic compound,it dephosphorylates extracellular AMP to adenosine. In some embodiments,a CD73 molecule dephosphorylates extracellular AMP to adenosineequivalent to, or at least, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95%of the rate of a naturally occurring CD73, e.g., the CD73 from which theCD73 molecule was derived. In some embodiments, a CD73 molecule has atleast 60, 70, 80, 90, 95, 99, or 100% sequence identity, or substantialsequence identity, with a naturally occurring CD73. Exemplary CD73sequences include GenBank AAH65937.1 5′-nucleotidase, ecto (CD73) [Homosapiens] or a mature form from the following sequence,

(SEQ ID NO: 2) MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKEPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNIVIGNLICDAMINNNLRHADETFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKFSTGSHCHGSFSLIFLSLWAVIFVLYQ.

In some embodiments, a CD73 molecule comprises a soluble form of CD73which can be shed from the membrane of endothelial cells by proteolyticcleavage or hydrolysis of the GPI anchor by shear stress see, e.g.,Reference: Yegutkin G, Bodin P, Burnstock G. Effect of shear stress onthe release of soluble ecto-enzymes ATPase and 5′-nucleotidase alongwith endogenous ATP from vascular endothelial cells. Br J Pharmacol2000; 129: 921-6. For CD73 function see Colgan et al., Physiologicalroles for ecto-5′-nucleotidase (CD73), Purinergic Signalling, June 2006,2:351.

Cell surface molecule binder, as that term is used herein, refers to amolecule, typically a polypeptide, that binds, e.g., specifically, to acell surface molecule on a cell, e.g., an immunosuppressive immune cell,e.g., a Treg. In some embodiments, the cell surface binder hassufficient sequence from a naturally occurring ligand of the cellsurface molecule, that it can specifically bind the cell surfacemolecule (a cell surface molecule ligand). In some embodiments, the cellsurface binding is an antibody molecule that binds, e.g., specificallybinds, the cell surface molecule.

Donor specific targeting moiety, as that term is used herein, refers toa moiety, e.g., an antibody molecule, that as a component of atherapeutic compound, localizes the therapeutic compound preferentiallyto an implanted donor tissue, as opposed to tissue of a recipient. As acomponent of a therapeutic compound, the donor specific targeting moietyprovides site-specific immune privilege for a transplant tissue, e.g.,an organ, from a donor.

In some embodiments, a donor specific targeting moiety it binds to theproduct, e.g., a polypeptide product, of an allele present at a locus,which allele is not present at the locus in the (recipient) subject. Insome embodiments, a donor specific targeting moiety binds to an epitopeon product, which epitope is not present in the (recipient) subject.

In some embodiments, a donor specific targeting moiety, as a componentof a therapeutic compound, preferentially binds to a donor target orantigen, e.g., has a binding affinity for the donor target that isgreater for donor antigen or tissue, e.g., at least 2, 4, 5, 10, 50,100, 500, 1,000, 5,000, or 10,000 fold greater, than its affinity forthan for subject antigen or tissue. In some embodiments, a donorspecific targeting moiety, has a binding affinity for a product of anallele of a locus present in donor tissue (but not present in thesubject) at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000fold greater, than its affinity for the product of the allele of thelocus present in the subject (which allele is not present in donortissue). Affinity of a therapeutic compound of which the donor specificmoiety is a component, can be measured in a cell suspension, e.g., theaffinity for suspended cells having the allele is compared with itsaffinity for suspended cells not having the allele. In some embodiments,the binding affinity for the donor allele cells is below 10 nM. In someembodiments, the binding affinity for the donor allele cells is below100 pM, 50 pM, or 10 pM.

In some embodiments, the specificity for a product of a donor allele issufficient that when the donor specific targeting moiety is coupled toan immune-down regulating effector: i) immune attack of the implantedtissue, e.g., as measured by histological inflammatory response,infiltrating T effector cells, or organ function, in the clinicalsetting—e.g. creatinine for the kidney, is substantially reduced, e.g.,as compared to what would be seen in an otherwise similar implant butlacking the donor specific targeting moiety is coupled to an immune-downregulating effector; and/or ii) immune function in the recipient,outside or away from the implanted tissue, is substantially maintained.In some embodiments, one or more of the following is seen: attherapeutic levels of therapeutic compound, peripheral blood lymphocytecounts are not substantially impacted, e.g., the level of T cells iswithin 25, 50, 75, 85, 90, or 95% of normal, the level of B cells iswithin 25, 50, 75, 85, 90, or 95% of normal, and/or the level ofgranuloctyes (PMNs) cells is within 25, 50, 75, 85, 90, or 95% ofnormal, or the level of monocytes is within 25, 50, 75, 85, 90, or 95%of normal; at therapeutic levels of therapeutic compound, the ex vivoproliferative function of PBMCs (peripheral blood mononuclear cells)against non-disease relevant antigens is substantially normal or iswithin 70, 80, or 90% of normal; at therapeutic levels of therapeuticcompound, the incidence or risk of risk of opportunistic infections andcancers associated with immunosuppression is not substantially increasedover normal; or at therapeutic levels of therapeutic compound, theincidence or risk of risk of opportunistic infections and cancersassociated with immunosuppression is substantially less than would beseen with standard of care, or non-targeted, immunosuppression. In someembodiments, the donor specific targeting moiety comprises an antibodymolecule, a target specific binding polypeptide, or a target ligandbinding molecule.

Effector, as that term is used herein, refers to an entity, e.g., a cellor molecule, e.g., a soluble or cell surface molecule, which mediates animmune response.

Effector ligand binding molecule, as used herein, refers to apolypeptide that has sufficient sequence from a naturally occurringcounter-ligand of an effector, that it can bind the effector withsufficient specificity that it can serve as an effectorbinding/modulating molecule. In some embodiments, it binds to effectorwith at least 10, 20, 30, 40, 50, 60, 70, 80, 90, or 95% of the affinityof the naturally occurring counter-ligand. In some embodiments, it hasat least 60, 70, 80, 90, 95, 99, or 100% sequence identity, orsubstantial sequence identity, with a naturally occurring counter-ligandfor the effector.

Effector specific binding polypeptide, as used herein, refers to apolypeptide that can bind with sufficient specificity that it can serveas an effector binding/modulating moiety. In some embodiments, aspecific binding polypeptide comprises a effector ligand bindingmolecule.

Elevated risk, as used herein, refers to the risk of a disorder in asubject, wherein the subject has one or more of a medical history of thedisorder or a symptom of the disorder, a biomarker associated with thedisorder or a symptom of the disorder, or a family history of thedisorder or a symptom of the disorder.

Functional antibody molecule to an effector or inhibitory immunecheckpoint molecule, as that term is used herein, refers to an antibodymolecule that when present as the ICIM binding/modulating moiety of amultimerized therapeutic compound, can bind and agonize the effector orinhibitory immune checkpoint molecule. In some embodiments, theanti-effector or inhibitory immune checkpoint molecule antibodymolecule, when binding as a monomer (or binding when the therapeuticcompound is not multimerized), to the effector or inhibitory immunecheckpoint molecule, does not antagonize, substantially antagonize,prevent binding, or prevent substantial binding, of an endogenouscounter ligand of the inhibitory immune checkpoint molecule molecule toinhibitory immune checkpoint molecule. In some embodiments, theanti-effector or inhibitory immune checkpoint molecule antibody moleculewhen binding as a monomer (or binding when the therapeutic compound isnot multimerized), to the inhibitory immune checkpoint molecule, doesnot agonize or substantially agonize, the effector or inhibitorymolecule.

ICIM binding/modulating moiety, as that term is used herein, refers toan effector binding/modulating moiety that, as part of a therapeuticcompound, binds and agonizes a cell surface inhibitory molecule, e.g.,an inhibitory immune checkpoint molecule, e.g., PD-1, or binds ormodulates cell signaling, e.g., binds a FCRL, e.g., FCRL1-6, or bindsand antagonizes a molecule that promotes immune function.

IIC binding/modulating moiety, as that term is used herein, refers to aneffector binding/modulating moiety that, as part of a therapeuticcompound, binds an immunosuppressive immune cell. In some embodiments,the IIC binding/modulating moiety increases the number or concentrationof an immunosuppressive immune cell at the binding site.

ICSM binding/modulating moiety, as that term is used herein, refers toan effector binding/modulating moiety that antagonizes an immunestimulatory effect of a stimulatory, e.g., co-stimulatory, binding pair.A stimulatory or co-stimulatory binding pair, as that term is usedherein, comprises two members, 1) a molecule on the surface of an immunecell; and 2) the binding partner for that cell molecule, which may be anadditional immune cell, or a non-immune cell. Ordinarily, upon bindingof one member to the other, assuming other requirements are met, themember on the immune cell surfaces stimulates the immune cell, e.g., acostimulatory molecule, and an immune response is promoted. Insituations where the costimulatory molecule and the costimulatorymolecule counterstructure are both expressed on immune cells,bi-directional activation of both cells may occur. In an embodiment anICSM binding/modulating moiety binds and antagonizes the immune cellexpressed member of a binding pair. For example, it binds andantagonizes OX40. In another embodiment, an ICSM binding/modulatingmoiety binds and antagonizes the member of the binding pair that itselfbinds the immune cell expressed member, e.g., it binds and antagonizesOX40L. In either case, inhibition of stimulation or co-stimulation of animmune cell is achieved. In an embodiment the ICSM binding/modulatingmoiety decreases the number or the activity of an immunostimulatingimmune cell at the binding site.

IL-2 mutein molecule, as that term is used herein, refers to an IL2variant that binds with high affinity to the CD25 (IL-2R alpha chain)and with low affinity to the other IL-2R signaling components CD122(IL-2R beta) and CD132 (IL-2R gamma). Such an IL-2 mutein moleculepreferentially activates Treg cells. In embodiments, either alone, or asa component of a therapeutic compound, an IL-2 mutein activates Tregs atleast 2, 5, 10, or 100 fold more than cytotoxic or effector T cells.Exemplary IL-2 mutein molecules are described in WO2010085495,WO2016/164937, US2014/0286898A1, WO2014153111A2, WO2010/085495,cytotoxic WO2016014428A2, WO2016025385A1, and US20060269515. Muteinsdisclosed in these references that include additional domains, e.g., anFc domain, or other domain for extension of half life can be used in thetherapeutic compounds and methods described herein without suchadditional domains. In another embodiment an IIC binding/modulatingmoiety comprises an IL-2 mutein, or active fragment thereof, coupled,e.g., fused, to another polypeptide, e.g., a polypeptide that extends invivo half life, e.g., an immunoglobulin constant region, or a multimeror dimer thereof, e.g., AMG 592. In an embodiment the therapeuticcompound comprises the IL-2 portion of AMG 592. In an embodiment thetherapeutic compound comprises the IL-2 portion but not theimmunoglobulin portion of AMG 592. In some embodiments, the mutein doesnot comprise a Fc region. For some IL-2 muteins, the muteins areengineered to contain a Fc region because such region has been shown toincrease the half-life of the mutein. In some embodiments, the extendedhalf-life is not necessary for the methods described and embodiedherein. In some embodiments, the Fc region that is fused with the IL-2mutein comprises a N297 mutations, such as, but not limited to, N297A.In some embodiments, the Fc region that is fused with the IL-2 muteindoes not comprise a N297 mutation, such as, but not limited to, N297A.

An “inhibitory immune checkpoint molecule ligand molecule,” as that termis used herein, refers to a polypeptide having sufficient inhibitoryimmune checkpoint molecule ligand sequence, e.g., in the case of a PD-L1molecule, sufficient PD-L1 sequence, that when present as an ICIMbinding/modulating moiety of a multimerized therapeutic compound, canbind and agonize its cognate inhibitory immune checkpoint molecule,e.g., again in the case of a PD-L1 molecule, PD-1.

In some embodiments, the inhibitory immune checkpoint molecule ligandmolecule, e.g., a PD-L1 molecule, when binding as a monomer (or bindingwhen the therapeutic compound is not multimerized), to its cognateligand, e.g., PD-1, does not antagonize or substantially antagonize, orprevent binding, or prevent substantial binding, of an endogenousinhibitory immune checkpoint molecule ligand to the inhibitory immunecheckpoint molecule. E.g., in the case of a PD-L1 molecule, the PD-L1molecule does not antagonize binding of endogenous PD-L1 to PD-1.

In some embodiments, the inhibitory immune checkpoint molecule ligandwhen binding as a monomer, to its cognate inhibitory immune checkpointmolecule does not agonize or substantially agonize the inhibitory immunecheckpoint molecule. By way of example, e.g., a PD-L1 molecule whenbinding to PD-1, does not agonize or substantially agonize PD-1. In someembodiments, an inhibitory immune checkpoint molecule ligand moleculehas at least 60, 70, 80, 90, 95, 99, or 100% sequence identity, orsubstantial sequence identity, with a naturally occurring inhibitoryimmune checkpoint molecule ligand.

Exemplary inhibitory immune checkpoint molecule ligand moleculesinclude: a PD-L1 molecule, which binds to inhibitory immune checkpointmolecule PD-1, and in embodiments has at least 60, 70, 80, 90, 95, 99,or 100% sequence identity, or substantial sequence identity, with anaturally occurring PD-L1, e.g., the PD-L1 molecule comprising thesequence of

(SEQ ID NO: 3) MIMEAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNERTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET,or an active fragment thereof; in some embodiments, the active fragmentcomprises residues 19 to 290 of the PD-L1 sequence; a HLA-G molecule,which binds to any of inhibitory immune checkpoint molecules KIR2DL4,LILRB1, and LILRB2, and in embodiments has at least 60, 70, 80, 90, 95,99, or 100% sequence identity, or substantial sequence identity, with anaturally occurring HLA-G. Exemplary HLA-G sequences include, e.g., amature form found in the sequence at GenBank P17693.1 RecName: Full=HLAclass I histocompatibility antigen, alpha chain G; AltName: Full=HLA Gantigen; AltName: Full=MHC class I antigen G; Flags: Precursor, or inthe sequence

(SEQ ID NO: 4) MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVEDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQ SSLPTIPIMGIVA.

Inhibitory molecule counter ligand molecule, as that term is usedherein, refers to a polypeptide having sufficient inhibitory moleculecounter ligand sequence such that when present as the ICIMbinding/modulating moiety of a multimerized therapeutic compound, canbind and agonize a cognate inhibitory molecule. In some embodiments, theinhibitory molecule counter ligand molecule, when binding as a monomer(or binding when the therapeutic compound is not multimerized), to theinhibitory molecule, does not antagonize, substantially antagonize,prevent binding, or prevent substantial binding, of an endogenouscounter ligand of the inhibitory molecule to the inhibitory molecule. Insome embodiments, the inhibitory molecule counter ligand molecule whenbinding as a monomer (or binding when the therapeutic compound is notmultimerized), to the inhibitory molecule, does not agonize orsubstantially agonize, the inhibitory molecule.

Sequence identity, percentage identity, and related terms, as thoseterms are used herein, refer to the relatedness of two sequences, e.g.,two nucleic acid sequences or two amino acid or polypeptide sequences.In the context of an amino acid sequence, the term “substantiallyidentical” is used herein to refer to a first amino acid that contains asufficient or minimum number of amino acid residues that are i)identical to, or ii) conservative substitutions of aligned amino acidresidues in a second amino acid sequence such that the first and secondamino acid sequences can have a common structural domain and/or commonfunctional activity. For example, amino acid sequences that contain acommon structural domain having at least about 85%, 90%. 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence, e.g., asequence provided herein.

In the context of nucleotide sequence, the term “substantiallyidentical” is used herein to refer to a first nucleic acid sequence thatcontains a sufficient or minimum number of nucleotides that areidentical to aligned nucleotides in a second nucleic acid sequence suchthat the first and second nucleotide sequences encode a polypeptidehaving common functional activity, or encode a common structuralpolypeptide domain or a common functional polypeptide activity. Forexample, nucleotide sequences having at least about 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a reference sequence,e.g., a sequence provided herein.

The term “functional variant” refers to polypeptides that have asubstantially identical amino acid sequence to the naturally-occurringsequence, or are encoded by a substantially identical nucleotidesequence, and are capable of having one or more activities of thenaturally-occurring sequence.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, 60%, and even more preferably at least 70%,80%, 90%, 100% of the length of the reference sequence. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”).

The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporatedinto the GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossum 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Aparticularly preferred set of parameters (and the one that should beused unless otherwise specified) are a Blossum 62 scoring matrix with agap penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller ((1989)CABIOS, 4:11-17) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and) (BLAST programs (version2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to for exampleany a nucleic acid sequence provided herein. BLAST protein searches canbe performed with the)(BLAST program, score=50, wordlength=3 to obtainamino acid sequences homologous to protein molecules provided herein. Toobtain gapped alignments for comparison purposes, Gapped BLAST can beutilized as described in Altschul et al., (1997) Nucleic Acids Res.25:3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)can be used. See http://www.ncbi.nlm.nih.gov.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: 1) low stringencyhybridization conditions in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); 2) medium stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 60° C.; 3) high stringency hybridization conditions in 6×SSC atabout 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 65°C.; and preferably 4) very high stringency hybridization conditions are0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washesat 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are thepreferred conditions and the ones that should be used unless otherwisespecified.

It is understood that the molecules and compounds of the presentembodiments may have additional conservative or non-essential amino acidsubstitutions, which do not have a substantial effect on theirfunctions.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof; amino acid analogs having variant side chains; and allstereoisomers of any of any of the foregoing. As used herein the term“amino acid” includes both the D- or L-optical isomers andpeptidomimetics. A “conservative amino acid substitution” is one inwhich the amino acid residue is replaced with an amino acid residuehaving a similar side chain. Families of amino acid residues havingsimilar side chains have been defined in the art. These families includeamino acids with basic side chains (e.g., lysine, arginine, histidine),acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polarside chains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). CD39 molecule, a CD73 molecule, a Cell surface moleculebinder, Donor specific targeting moiety Effector ligand bindingmolecule, ICIM binding/modulating moiety IIC binding/modulating moiety,an inhibitory immune checkpoint molecule ligand molecule, Inhibitorymolecule counter ligand molecule, SM binding/modulating moiety, or ICSMbinding/modulating moiety.

SM binding/modulating moiety, as that term is used herein, refers to aneffector binding/modulating moiety that, as part of a therapeuticcompound, promotes an immuno-suppressive local microenvironment, e.g.,by providing in the proximity of the target, a substance that inhibitsor minimizes attack by the immune system of the target. In someembodiments, the SM binding/modulating moiety comprises, or binds, amolecule that inhibits or minimizes attack by the immune system of thetarget. In some embodiments, a therapeutic compound comprises an SMbinding/modulating moiety that binds and accumulates a solublesubstance, e.g., an endogenous or exogenous substance, havingimmunosuppressive function. In some embodiments, a therapeutic compoundcomprises an SM binding/modulating moiety that binds and inhibits,sequesters, degrades or otherwise neutralizes a substance, e.g., asoluble substance, typically and endogenous soluble substance, thatpromotes immune attack. In some embodiments, a therapeutic compoundcomprises an SM binding/modulating moiety that comprises animmune-suppressive substance, e.g. a fragment of protein known to beimmunosuppressive. By way of example, an effector molecule bindingmoiety that binds, or comprises, a substance e.g., a CD39 molecule or aCD73 molecule, that depletes a component, that promotes immune effectorcell function, e.g., ATP or AMP.

Specific targeting moiety, as that term is used herein, refers to donorspecific targeting moiety or a tissue specific targeting moiety.

Subject, as that term is used herein, refers to a mammalian subject,e.g., a human subject. In some embodiments, the subject is a non-humanmammal, e.g., a horse, dog, cat, cow, goat, or pig.

Target ligand binding molecule, as used herein, refers to a polypeptidethat has sufficient sequence from a naturally occurring counter-ligandof a target ligand that it can bind the target ligand on a target tissue(e.g., donor tissue or subject target tissue) with sufficientspecificity that it can serve as a specific targeting moiety. In someembodiments, it binds to target tissue or cells with at least 10, 20,30, 40, 50, 60, 70, 80, 90, or 95% of the affinity of the naturallyoccurring counter-ligand. In some embodiments, it has at least 60, 70,80, 90, 95, 99, or 100% sequence identity, or substantial sequenceidentity, with a naturally occurring counter-ligand for the targetligand.

Target site, as that term is used herein, refers to a site whichcontains the entity, e.g., epitope, bound by a targeting moiety. In someembodiments, the target site is the site at which immune privilege isestablished.

Tissue specific targeting moiety, as that term is used herein, refers toa moiety, e.g., an antibody molecule, that as a component of atherapeutic molecule, localizes the therapeutic molecule preferentiallyto a target tissue, as opposed to other tissue of a subject. As acomponent of a therapeutic compound, the tissue specific targetingmoiety provides site-specific immune privilege for a target tissue,e.g., an organ or tissue undergoing or at risk for autoimmune attack. Insome embodiments, a tissue specific targeting moiety binds to a product,e.g., a polypeptide product, which is not present outside the targettissue, or is present at sufficiently low levels that, at therapeuticconcentrations of therapeutic molecule, unacceptable levels of immunesuppression are absent or substantially absent. In some embodiments, atissue specific targeting moiety binds to an epitope, which epitope isnot present outside, or not substantially present outside, the targetsite.

In some embodiments, a tissue specific targeting moiety, as a componentof a therapeutic compound, preferentially binds to a target tissue ortarget tissue antigen, e.g., has a binding affinity for the targettissue or antigen that is greater for target antigen or tissue, e.g., atleast 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000 fold greater,than its affinity for than for non-target tissue or antigen presentoutside the target tissue. Affinity of a therapeutic compound of whichthe tissue specific moiety is a component, can be measured in a cellsuspension, e.g., the affinity for suspended cells having the targetantigen is compared with its affinity for suspended cells not having thetarget antigen. In some embodiments, the binding affinity for the targetantigen bearing cells is below 10 nM.

In some embodiments, the binding affinity for the target antigen bearingcells is below 100 pM, 50 pM, or 10 pM. In some embodiments, thespecificity for a target antigen is sufficient, that when the tissuespecific targeting moiety is coupled to an immune-down regulatingeffector: i) immune attack of the target tissue, e.g., as measured byhistological inflammatory response, infiltrating T effector cells, ororgan function, in the clinical setting—e.g. creatinine for kidney, issubstantially reduced, e.g., as compared to what would be seen in anotherwise similar implant but lacking the tissue specific targetingmoiety is coupled to an immune-down regulating effector; and/or ii)immune function in the recipient, outside or away from the targettissue, is substantially maintained.

In some embodiments, one or more of the following is seen: attherapeutic levels of therapeutic compound, peripheral blood lymphocytecounts are not substantially impacted, e.g., the level of T cells iswithin 25, 50, 75, 85, 90, or 95% of normal, the level of B cells iswithin 25, 50, 75, 85, 90, or 95% of normal, and/or the level ofgranulocytes (PMNs) cells is within 25, 50, 75, 85, 90, or 95% ofnormal, or the level of monocytes is within 25, 50, 75, 85, 90, or 95%of normal 1; at therapeutic levels of therapeutic compound, the ex vivoproliferative function of PBMCs (peripheral blood mononuclear cells)against non-disease relevant antigens is substantially normal or iswithin 70, 80, or 90% of normal; at therapeutic levels of therapeuticcompound, the incidence or risk of risk of opportunistic infections andcancers associated with immunosuppression is not substantially increasedover normal; or at therapeutic levels of therapeutic compound, theincidence or risk of risk of opportunistic infections and cancersassociated with immunosuppression is substantially less than would beseen with standard of care, or non-targeted, immunosuppression. In someembodiments, the tissue specific targeting moiety comprises an antibodymolecule. In some embodiments, the donor specific targeting moietycomprises an antibody molecule, a target specific binding polypeptide,or a target ligand binding molecule. In some embodiments, the tissuespecific targeting moiety binds a product, or a site on a product, thatis present or expressed exclusively, or substantially exclusively, ontarget tissue.

ICIM Binding/Modulating Moieties: Effector Binding/Modulating Moietiesthat Bind Inhibitory Receptors

Methods and compounds described herein provide for a therapeuticcompound having an effector binding/modulating moiety comprising an ICIMbinding/modulating moiety, that directly binds and activates aninhibitory receptor on the surface of an immune cell, e.g., to reduce oreliminate, or substantially eliminate, the ability of the immune cell tomediate immune attack. Coupling of the ICIM binding/modulating moiety toa targeting entity, promotes site-specific or local down regulation ofthe immune cell response, e.g., confined substantially to the locationshaving binding sites for the targeting moiety. Thus, normal systemicimmune function is substantially retained. In some embodiments, an ICIMbinding/modulating moiety comprises an inhibitory immune checkpointmolecule counter ligand molecule, e.g., a natural ligand, or fragment ofa natural ligand (e.g., PD-L1 or HLA-G) of the inhibitory immunecheckpoint molecule. In some embodiments, an ICIM binding/modulatingmoiety comprises a functional antibody molecule, e.g., a functionalantibody molecule comprising an scFv binding domain, that engagesinhibitory immune checkpoint molecule.

In some embodiments, the ICIM binding/modulating moiety, comprising,e.g., a functional antibody molecule, or inhibitory immune checkpointmolecule ligand molecule, binds the inhibitory receptor but does notprevent binding of a natural ligand of the inhibitory receptor to theinhibitory receptor. In embodiments a format is used wherein a targetingmoiety is coupled, e.g., fused, to an ICIM binding/modulating moiety,comprising, e.g., an scFv domain, and configured so that upon binding ofan inhibitory receptor while in solution (e.g., in blood or lymph) (andpresumably in a monomeric format), the therapeutic molecule: i) fails toagonize, or fails to substantially agonize (e.g., agonizes at less than30, 20, 15, 10, or 5% of the level seen with a full agonizing molecule)the inhibitory receptor on the immune cell; and/or ii) fails toantagonize, or fails to substantially antagonize (e.g., antagonizes atless than 30, 20, 15, 10, or 5% of the level seen with a fullantagonizing molecule) the inhibitory receptor on the immune cell. Acandidate molecule can be evaluated for its ability to agonize or notagonize by its ability to either increase or decrease the immuneresponse in an in vitro cell based assay wherein the target is notexpressed, e.g., using an MLR-based assay (mixed lymphocyte reaction).

In some embodiments, candidate ICIM binding/modulating moieties canreduce, completely or substantially eliminate systemic immunosuppressionand systemic immune activation. In some embodiments, the targetingdomain of the therapeutic compound, when bound to target, will serve tocluster or multimerize the therapeutic compound on the surface of thetissue desiring immune protection. In some embodiments, the ICIMbinding/modulating moiety, e.g., an ICIM binding/modulating moietycomprising a scFv domain, requires a clustered or multimeric state to beable to deliver an agonistic and immunosuppressive signal, orsubstantial levels of such signal, to local immune cells. This type oftherapeutic can, for example, provide to a local immune suppressionwhilst leaving the systemic immune system unperturbed or substantiallyunperturbed. That is, the immune suppression is localized to where thesuppression is needed as opposed to being systemic and not localized toa particular area or tissue type.

In some embodiments, upon binding to the target e.g., a target organ,tissue or cell type, the therapeutic compound coats the target, e.g.,target organ, tissue or cell type. When circulating lymphocytes attemptto engage and destroy the target, this therapeutic will provide an ‘off’signal only at, or to a greater extent at, the site of therapeuticcompound accumulation.

A candidate therapeutic compound can be evaluated for the ability tobind, e.g., specifically bind, its target, e.g., by ELISA, a cell basedassay, or surface plasmon resonance. by. This property should generallybe maximized, as it mediates the site-specificity and local nature ofthe immune privilege. A candidate therapeutic compound can be evaluatedfor the ability to down regulate an immune cell when bound to target,e.g., by a cell based activity assay. This property should generally bemaximized, as it mediates the site-specificity and local nature of theimmune privilege. The level of down regulation effected by a candidatetherapeutic compound in monomeric (or non-bound) form can be evaluated,e.g., by a cell based activity assay. This property should generally beminimized, as could mediate systemic down regulation of the immunesystem. The level of antagonism of a cell surface inhibitory molecule,e.g., an inhibitory immune checkpoint molecule, effected by a candidatetherapeutic compound in monomeric (or non-bound) form can be evaluated,e.g.,by, e.g., by a cell based activity assay. This property shouldgenerally be minimized, as could mediate systemic unwanted activation ofthe immune system. Generally, the properties should be selected andbalanced to produce a sufficiently robust site specific immune privilegewithout unacceptable levels of non-site specific agonism or antagonismof the inhibitory immune checkpoint molecule.

Exemplary Inhibitory Immune Checkpoint Molecules

Exemplary inhibitory molecules (e.g., an inhibitory immune checkpointmolecule) (together with their counter ligands) can be found in Table 1.This table lists molecules to which exemplary ICIM binding moieties canbind.

TABLE 1 Cell surface inhibitory molecules, e.g., inhibitory immunecheckpoint molecules (column A), counter ligands (column B) and celltypes affected (column C). THE PD-Ll/PD-1 PATHWAY A B C PD-1 PD-L1,PD-L2 T cells, B cells Alkaline phosphatase B7-H3 Unknown T cells B7-H4Neuropilin 1, T cells neuropilin 2, Plexin4A BTLA HVEM T cells, B cellsCTLA-4 CD80, CD86 T cells IDO1 Tryptophan Lymphocytes TDO2 TryptophanLymphocytes KIR2DL1, HLA MHC class I NK cells KIR2DL2/3, KIR3DL1,KIR3DL2 LAG3 HLA MHC class II T cells TIM-3 Galectin-9 T cells VISTAUnknown T cells, myeloid cells TIGIT CD155 T cells KIR2DL4 HLA-G NKcells LILRB1 HLA-G T cells, NK cells, B cells, monocytes, dendriticcells LILRB2 HLA-G Monocytes, dendritic cells, neutrophils, some tumorcells NKG2A nonclassical MHC T cells, NK cells glycoproteins class IFCRL1-6 FCRL1-2 not known B cells FCRL4 = IgA FCRL5 = IgG FCRL6 = MHCClass II BUTYROPHILINS, Modulation of immune cells for example BTN1A1,BTN2A2, BTNL2, BTNL1, BTNL8

Programmed cell death protein 1, (often referred to as PD-1) is a cellsurface receptor that belongs to the immunoglobulin superfamily. PD-1 isexpressed on T cells and other cell types including, but not limited to,B cells, myeloid cells, dendritic cells, monocytes, T regulatory cells,iNK T cells. PD-1 binds two ligands, PD-L1 and PD-L2, and is aninhibitory immune checkpoint molecule. Engagement with a cognate ligand,PD-L1 or PD-L2, in the context of engagement of antigen loaded MCH withthe T Cell Receptor on a T cell minimizes or prevents the activation andfunction of T cells. The inhibitory effect of PD-1 can include bothpromoting apoptosis (programmed cell death) in antigen specific T-cellsin lymph nodes and reducing apoptosis in regulatory T cells (suppressorT cells).

In some embodiments, a therapeutic compound comprises an ICIMbinding/modulating moiety which agonizes PD-1 inhibition. An ICIMbinding/modulating moiety can include an inhibitory molecule counterligand molecule, e.g., comprising a fragment of a ligand of PD-1 (e.g.,a fragment of PD-L1 or PD-L2) or another moiety, e.g., a functionalantibody molecule, comprising, e.g., an scFv domain that binds PD-1.

In some embodiments, a therapeutic compound comprises a targeting moietythat is preferentially binds a donor antigen not present in, present insubstantially lower levels in the subject, e.g., a donor antigen fromTable 2, and is localized to donor graft tissue in a subject. In someembodiments, it does not bind, or does not substantially bind, othertissues. In some embodiments, a therapeutic compound can include atargeting moiety that is specific for HLA-A2 and specifically bindsdonor allograft tissue but does not bind, or does not substantiallybind, host tissues. In some embodiments, the therapeutic compoundcomprises an ICIM binding/modulating moiety, e.g., an inhibitorymolecule counter ligand molecule, e.g., comprising a fragment of aligand of PD-1 (e.g., a fragment of PD-L1 or PD-L2) or another moiety,e.g., a functional antibody molecule, comprising, e.g., an scFv domainthat binds PD-1, such that the therapeutic compound, e.g., when bound totarget, activates PD-1. The therapeutic compound targets an allograftand provides local immune privilege to the allograft.

In some embodiments, a therapeutic compound comprises a targeting moietythat is preferentially binds to an antigen of Table 3, and is localizedto the target in a subject, e.g., a subject having an autoimmunedisorder, e.g., an autoimmune disorder of Table 3. In some embodiments,it does not bind, or does not substantially bind, other tissues. In someembodiments, the therapeutic compound comprises an ICIMbinding/modulating moiety, e.g., an inhibitory molecule counter ligandmolecule, e.g., comprising a fragment of a ligand of PD-1 (e.g., afragment of PD-L1 or PD-L2) or another moiety, e.g., a functionalantibody molecule, comprising, e.g., an scFv domain that binds PD-1,such that the therapeutic compound, e.g., when bound to target,activates PD-1. The therapeutic compound targets a tissue subject toautoimmune attack and provides local immune privilege to the tissue.

PD-L1 and PDL2, or polypeptides derived therefrom, can provide candidateICIM binding moieties. However, in monomer form, e.g., when thetherapeutic compound is circulating in blood or lymph, this moleculecould have an undesired effect of antagonizing the PD-L1/PD-1 pathway,and may only agonize the PD-1 pathway when clustered or multimerized onthe surface of a target, e.g., a target organ. In some embodiments, atherapeutic compound comprises an ICIM binding/modulating moietycomprising a functional antibody molecule, e.g., a scFv domain, that isinert, or substantially inert, to the PD-1 pathway in a soluble form butwhich agonizes and drives an inhibitory signal when multimerized (by thetargeting moiety) on the surface of a tissue.

The HLA-G: KIR2DL4/LILRB1/LILRB2 Pathway

KIR2DL4, LILRB1, and LILRB2 are inhibitory molecules found on T cells,NK cells, and myeloid cells. HLA-G is a counter ligand for each.

KIR2DL4 is also known as CD158D, G9P, KIR-103AS, KIR103, KIR103AS, KIR,KIR-2DL4, killer cell immunoglobulin like receptor, and two Ig domainsand long cytoplasmic tail 4. LILRB1 is also known as LILRB1, CD85J,ILT-2, ILT2, LIR-1, LIR1, MIR-7, MIR7, PIR-B, PIRB, leukocyteimmunoglobulin like receptor B1. LILRB2 is also known as CD85D, ILT-4,LIR-2, LIR2, MIR-10, MIR10, and ILT4.

A therapeutic compound comprising an HLA-G molecule can be used toprovide inhibitory signals to an immune cell comprising any of KIR2DL4,LILRB1, and LILRB2, e.g., with multimerized therapeutic compoundmolecules comprising an HLA-G molecule and thus provide site-specificimmune privilege.

A therapeutic compound comprising an agonistic anti-KIR2DL4,anti-LILRB1, or anti-LILRB2 antibody molecule can be used to provideinhibitory signals to an immune cell comprising any of KIR2DL4, LILRB1,and LILRB2.

HLA-G only delivers an inhibitory signal when multimerized, for example,when expressed on the surface of a cell or when conjugated to thesurface of a bead. In embodiments, a therapeutic compound comprising anHLA-G molecule which therapeutic compound does not multimerize insolution (or does not multimerize sufficiently to result in significantlevels of inhibitory molecule agonization), is provided. The use ofHLA-G molecules that minimize mulitmerization in solution will minimizesystemic agonization of immune cells and unwanted immune suppression.

While not wishing to be bound by theory it is believed that HLA-G is noteffective in down regulation unless multimerized, that binding of thetherapeutic compound to target, through the targeting moiety,multimerizes the ICIM binding entity, and that the multimerized ICIMbinding entity, binds and clusters inhibitory molecules on the surfaceof an immune cell, thus mediating a negative signal that down regulatesthe immune cell. Thus, infiltrating immune cells attempting to damagethe target tissue, including antigen presenting cells and other myeloidcells, NK cells and T cells, are down regulated.

While HLA-G molecules minimize antagonism when in monomeric form aredesirable, the redundancy of LILRB1 and LILRB2 will minimize, the impacton systemic even with some monomeric antagonism.

In some embodiments, the therapeutic compound comprises an ICIMbinding/modulating moiety that comprises a HLA-G molecule, e.g., anB2M-free isoform (e.g., HLA-G5), see Carosella et al., Advances inImmunology, 2015, 127:33. In a B2M-free format, HLA-G preferentiallybinds LILRB2.

Suitable sequences for the construction of HLA-G molecules includeGenBank P17693.1 RecName: Full=HLA class I histocompatibility antigen,alpha chain G; AltName: Full=HLA G antigen; AltName: Full=MHC class Iantigen G; Flags: Precursor, or

(SEQ ID NO: 5) MVVMAPRTLFLLLSGALTLTETWAGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD.A candidate HLA-G molecule can be tested for suitability for use inmethods and compounds, e.g., by methods analogous to those described in“Synthetic HLA-G proteins for therapeutic use in transplantation,”LeMaoult et al., 2013 The FASEB Journal 27:3643.

In some embodiments, a therapeutic compound comprises a targeting moietythat is preferentially binds a donor antigen not present in, present insubstantially lower levels in the subject, e.g., a donor antigen fromTable 2, and is localized to donor graft tissue in a subject. In someembodiments, it does not bind, or does not substantially bind, othertissues. In some embodiments, a therapeutic compound can include atargeting moiety that is specific for HLA-A2 and specifically binds adonor allograft but does not bind host tissues and is combined with anICIM binding/modulating moiety that comprises a HLA-G molecule thatbinds KIR2DL4, LILRB1, or LILRB2, such that the therapeutic compound,e.g., when bound to target, activates KIR2DL4, LILRB1, or LILRB2. Thetherapeutic compound targets an allograft and provides local immuneprivilege to the allograft.

In some embodiments, a therapeutic compound comprises a targeting moietythat is preferentially binds a tissue specific antigen, e.g., an antigenfrom Table 3, and is localized to the target site in a subject, e.g., asubject having an autoimmune disorder, e.g., an anutoimmune disorderfrom Table 3. In some embodiments, it does not bind, or does notsubstantially bind, other tissues. In embodiments the therapeuticcompound comprises an ICIM binding/modulating moiety that comprises aHLA-G molecule binds KIR2DL4, LILRB1, or LILRB2, such that thetherapeutic compound, e.g., when bound to target, activates KIR2DL4,LILRB1, or LILRB2. The therapeutic compound targets an tissue subject toautoimmune attack and provides local immune privilege to the tissue.

It is likely possible to engineer a stable and soluble HLA-G-B2M fusionprotein that can also bind LILRB1. For example, the crystal structure ofHLA-G was determined using HLA-G/B2M monomers (Clements et al. 2005 PNAS102:3360)

FCRL Family

FCRL1-6 generally inhibit B cell activation or function. These type 1transmembrane glycoproteins are composed of different combinations of 5types of immunoglobulin-like domains, with each protein consisting of 3to 9 domains, and no individual domain type conserved throughout all ofthe FCRL proteins. In general, FCRL expression is restricted tolymphocytes, with the primary expression in B-lymphocytes. Generally,FCRLs function to repress B-cell activation.

An ICIM binding/modulating moiety can comprise an agonistic anti-BCMAantibody molecule. In some embodiments, the therapeutic compoundcomprises an anti-FCRL antibody molecule and an anti-B cell receptor(BCR) antibody molecule. While not wishing to be bound be theory isbelieved that a therapeutic compound comprising anti-body molecules ofboth specificities will bring the FCRL into close proximity with the BCRand inhibit BCR signaling.

Butyrophilins and Butyrophilin-Like Molecules

Effector binding/modulating moiety can comprise an agonist or antagonistof a butyrophilin. In some embodiments, an effector binding/modulatingmoiety an agonistic or functional BTN1A1 molecule, BTN2A2 molecule,BTNL2 molecule, or BTNL1 molecule.

A functional BTNXi molecule (where Xi=1A1, 2A2, L2 or L1), as that termas used herein, refers to a polypeptide having sufficient BTNXi sequencethat, as part of a therapeutic compound, it inhibits T cells. In someembodiments, a BTNXi molecule has at least 60, 70, 80, 90, 95, 99, or100% sequence identity, or substantial sequence identity, with anaturally occurring butyrophilin or butyrophilin-like molecule.

In some embodiments, an effector binding/modulating moiety anantagonistic BTNL8 molecule.

An antagonistic BTNL8 molecule, as that term as used herein, refers to apolypeptide having sufficient BTNL8 sequence that, as part of atherapeutic compound, it inhibits the activation, proliferation, orsecretion of cytokine by a resting T cell. In some embodiments, a BTNL8molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequence identity,or substantial sequence identity, with a naturally occurringbutyrophilin.

IIC Binding/Modulating Moieties: Effector Binding/Modulating Moietiesthat Recruit Immunosuppressive T Cells

In some embodiments, a therapeutic compound comprises an effectorbinding/modulating moiety, e.g., an IIC binding/modulating moiety, thatbinds, activates, or retains immunosuppressive cells, e.g.,immunosuppressive T cells, at the site mediated by the targeting moiety,providing site-specific immune privilege. The IIC binding/modulatingmoiety, e.g., an IIC binding/modulating moiety comprising an antibodymolecule, comprising, e.g., an scFv binding domain, bindsimmunosuppressive cell types, e.g., Tregs, e.g., Foxp3+CD25+ Tregs.Organ, tissue or specific cell type tolerance is associated with anoverwhelming increase of Tregs proximal and infiltrating the targetorgan; in embodiments, the methods and compounds described hereinsynthetically re-create and mimic this physiological state. Uponaccumulation of Tregs, an immunosuppressive microenvironment is createdthat serves to protect the organ of interest from the immune system.

GARP-Binders as a Treg and TGFB Targeting Molecule

GARP is a membrane protein receptor for latent TGF-beta expressed on thesurface of activated Tregs (Tran et al. 2009 PNAS 106:13445 and Wang etal. 2009 PNAS 106:13439). In some embodiments, a therapeutic compoundcomprises an IIC binding entity that binds one or both of soluble GARPand GARP-expressing cells, such as activated human Tregs, and atargeting moiety that targets the therapeutic compound to the targettissue of interest. IIC binding/modulating moieties that comprises aGARP-Binder include, e.g., an IIC binding/modulating moiety thatcomprises an anti-GARP antibody molecule, e.g., an anti-GARP scFvdomain. While not wishing to be bound by theory, it is believed that thetherapeutic compound that comprises a GARP binder effects accumulationof GARP-expressing Tregs at the site targeted by the targeting moiety ofthe therapeutic compound, e.g., a transplant or site of organ injury.Again, while not wishing to be bound by theory, it is believed that atherapeutic compound that comprises a GARP binder effects can alsoeffect accumulation of soluble GARP at site of organ injury, which willserve to bind and activate TGFB1, an immuno-suppressive cytokine, in alocal manner (Fridrich et al. 2016 PLoS One 11:e0153290; doi:10.1371/journal.pone.0153290 and Hahn et al. 2013 Blood 15:1182). Thus,an effector binding/modulating moiety that comprises a GARP binder canact as either a IIC binding/modulating moiety or an SMbinding/modulating moiety.

CTLA4 as a Treg Targeting and T Effector Cell Silencing Molecule

In some embodiments, an effector binding/modulating moiety, e.g.,comprises an antibody molecule, e.g., an scFv domain, that binds CTLA4expressed on the surface of Tregs. The therapeutic molecule accumulatesor retains CTLA4+ Tregs at the target site, with local immunosuppressionthe consequence.

Though expressed more highly on Tregs, CTLA4 is also expressed onactivated T cells. A therapeutic compound comprising an effectorbinding/modulating moiety, e.g., an anti-CTLA4 antibody, or a functionalanti-CTLA4 antibody, can down regulate the CTLA4 expressing T cell.Thus, in a therapeutic compound comprising an effectorbinding/modulating moiety that binds CTLA4, the effector moiety can alsoact as an ICIM binding/modulating moiety.

In some embodiments, the anti-CTLA4 binder is neither antagonizing oragonizing when in monomeric format, and is only agonizing when clusteredor multimerized upon binding to the target.

While not wishing to be bound by theory it is believed that the bindingof the therapeutic compound, via the targeting moiety, to the target,effects multimerization of therapeutic compound. In the case of memoryand activated T cells, CTLA4 bound by the effector binding/modulatingmoiety of the therapeutic compound, is clustered, and an inhibitorysignal by engagement of CTLA4 expressed by memory and activated T cells

In some embodiments, the anti-CTLA4 binder is neither antagonizing oragonizing when in monomeric format, and is only agonizing when clusteredor multimerized upon binding to the target.

IL-2 Mutein Molecules: IL2 Receptor Binders that Activate Tregs

IL-2 mutein molecules that preferentially expand or stimulate Treg cells(over cytotoxic T cells) can be used as an IIC binding/modulatingmoiety.

In some embodiments, IIC binding/modulating moiety comprises a IL-2mutein molecule. As used herein, the term “IL-2 mutein molecule” or“IL-2 mutein” refers to an IL-2 variant that preferentially activatesTreg cells. In some embodiments, either alone, or as a component of atherapeutic compound, an IL-2 mutein molecule activates Tregs at least2, 5, 10, or 100 fold more than cytotoxic T cells. A suitable assay forevaluating preferential activation of Treg cells can be found in U.S.Pat. No. 9,580,486 at, for example, Examples 2 and 3, or in WO2016014428at, for example, Examples 3, 4, and 5, each of which is incorporated byreference in its entirety. The sequence of mature IL-2 is

(SEQ ID NO: 6) APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT (mature IL-2 sequence)The immature sequence of IL-2 can be represented by

(SEQ ID NO: 15) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT.

In some embodiments, an IIC binding/modulating moiety comprises an IL-2mutein, or active fragment thereof, coupled, e.g., fused, to anotherpolypeptide, e.g., a polypeptide that extends in vivo half life, e.g.,an immunoglobulin constant region, or a multimer or dimer thereof.

An IL-2 mutein molecule can be prepared by mutating one or more of theresidues of IL-2. Non-limiting examples of IL-2-muteins can be found inWO2016/164937, U.S. Pat. Nos. 9,580,486, 7,105,653, 9,616,105,9,428,567, US2017/0051029, US2014/0286898A1, WO2014153111A2,WO2010/085495, WO2016014428A2, WO2016025385A1, and US20060269515, eachof which are incorporated by reference in its entirety.

In some embodiments, the alanine at position 1 of the sequence above isdeleted. In some embodiments, the IL-2 mutein molecule comprises aserine substituted for cysteine at position 125 of the mature IL-2sequence. Other combinations of mutations and substitutions that areIL-2 mutein molecules are described in US20060269515, which isincorporated by reference in its entirety. In some embodiments, thecysteine at position 125 is also substituted with a valine or alanine.In some embodiments, the IL-2 mutein molecule comprises a V91Ksubstitution. In some embodiments, the IL-2 mutein molecule comprises aN88D substitution. In some embodiments, the IL-2 mutein moleculecomprises a N88R substitution. In some embodiments, the IL-2 muteinmolecule comprises a substitution of H16E, D84K, V91N, N88D, V91K, orV91R, any combinations thereof. In some embodiments, these IL-2 muteinmolecules also comprise a substitution at position 125 as describedherein. In some embodiments, the IL-2 mutein molecule comprises one ormore substitutions selected from the group consisting of: T3N, T3A,L12G, L12K, L12Q, L12S, Q13G, E15A, E15G, E15S, H16A, H16D, H16G, H16K,H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D, L19E, L19G, L19N,L19R, L19S, L19T, L19V, D20A, D20E, D20H, D20I, D20Y, D20F, D20G, D20T,D20W, M23R, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84QD84R, D84S, D84T, S87R, N88A, N88D, N88E, N88I, N88F, N88G, N88M, N88R,N88S, N88V, N88W, V91D, V91E, V91G, V91S, I92K, I92R, E95G, and Q126. Insome embodiments, the amino acid sequence of the IL-2 mutein moleculediffers from the amino acid sequence set forth in mature IL-2 sequencewith a C125A or C125S substitution and with one substitution selectedfrom T3N, T3A, L12G, L12K, L12Q L12S, Q13G, E15A, E15G, E15S, H16A,H16D, H16G, H16K, H16M, H16N, H16R, H16S, H16T, H16V, H16Y, L19A, L19D,L19E, L19G, L19N, L19R, L19S, L19T, L19V, D20A, D20E, D20F, D20G, D20T,D20W, M23R, R81A, R81G, R81S, R81T, D84A, D84E, D84G, D84I, D84M, D84Q,D84R, D84S, D84T, S87R, N88A, N88D, N88E, N88F, N88I, N88G, N88M, N88R,N88S, N88V, N88W, V91D, V91E, V91G, V91S, I92K, I92R, E95G, Q126I,Q126L, and Q126F. In some embodiments, the IL-2 mutein molecule differsfrom the amino acid sequence set forth in mature IL-2 sequence with aC125A or C125S substitution and with one substitution selected fromD20H, D20I, D20Y, D20E, D20G, D20W, D84A, D84S, H16D, H16G, H16K, H16R,H16T, H16V, I92K, I92R, L12K, L19D, L19N, L19T, N88D, N88R, N88S, V91D,V91G, V91K, and V91S. In some embodiments, the IL-2 mutein comprisesN88R and/or D20H mutations.

In some embodiments, the IL-2 mutein molecule comprises a mutation inthe polypeptide sequence at a position selected from the groupconsisting of amino acid 30, amino acid 31, amino acid 35, amino acid69, and amino acid 74. In some embodiments, the mutation at position 30is N30S. In some embodiments, the mutation at position 31 is Y31H. Insome embodiments, the mutation at position 35 is K35R. In someembodiments, the mutation at position 69 is V69A. In some embodiments,the mutation at position 74 is Q74P. In some embodiments, the muteincomprises a V69A mutation, a Q74P mutation, a N88D or N88R mutation, andone or more of L53I, L56I, L80I, or L118I mutations. In someembodiments, the mutein comprises a V69A mutation, a Q74P mutation, aN88D or N88R mutation, and a L to I mutation selected from the groupconsisting of: L53I, L56I, L80I, and L118I mutation. In someembodiments, the IL-2 mutein comprises a V69A, a Q74P, a N88D or N88Rmutation, and a L53I mutation. In some embodiments, the IL-2 muteincomprises a V69A, a Q74P, a N88D or N88R mutation, and a L56I mutation.In some embodiments, the IL-2 mutein comprises a V69A, a Q74P, a N88D orN88R mutation, and a L80I mutation. In some embodiments, the IL-2 muteincomprises a V69A, a Q74P, a N88D or N88R mutation, and a L118I mutation.As provided for herein, the muteins can also comprise a C125A or C125Smutation.

In some embodiments, the IL-2 mutein molecule comprises a substitutionselected from the group consisting of: N88R, N88I, N88G, D20H, D109C,Q126L, Q126F, D84G, or D84I relative to mature human IL-2 sequenceprovided above. In some embodiments, the IL-2 mutein molecule comprisesa substitution of D109C and one or both of a N88R substitution and aC125S substitution. In some embodiments, the cysteine that is in theIL-2 mutein molecule at position 109 is linked to a polyethylene glycolmoiety, wherein the polyethylene glycol moiety has a molecular weight ofbetween 5 and 40 kDa.

In some embodiments, any of the substitutions described herein arecombined with a substitution at position 125. The substitution can be aC125S, C125A, or C125V substitution.

In addition to the substitutions or mutations described herein, in someembodiments, the IL-2 mutein has a substitution/mutation at one or moreof positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 15 orpositions at one or more of positions 53, 56, 80, or 118 that correspondto SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises amutation at positions 73 and 76; 73 and 100; 73 and 138; 76 and 100; 76and 138; 100 and 138; 73, 76, and 100; 73, 76, and 138; 73, 100, and138; 76, 100 and 138; or at each of 73, 76, 100, and 138 that correspondto SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises amutation at positions 53 and 56; 53 and 80; 53 and 118; 56 and 80; 56and 118; 80 and 118; 53, 56, and 80; 53, 56, and 118; 53, 80, and 118;56, 80 and 118; or at each of 53, 56, 80, and 118 that correspond to SEQID NO: 6. As the IL-2 can be fused or tethered to other proteins, asused herein, the term corresponds to as reference to a SEQ ID Nos: 6 or15 refer to how the sequences would align with default settings foralignment software, such as can be used with the NCBI website. In someembodiments, the mutation is leucine to isoleucine. Thus, the IL-2mutein can comprise one more isoleucines at positions 73, 76, 100, or138 that correspond to SEQ ID NO: 15 or positions at one or more ofpositions 53, 56, 80, or 118 that correspond to SEQ ID NO: 6. In someembodiments, the mutein comprises a mutation at L53 that correspond toSEQ ID NO: 6. In some embodiments, the mutein comprises a mutation atL56 that correspond to SEQ ID NO: 6. In some embodiments, the muteincomprises a mutation at L80 that correspond to SEQ ID NO: 6. In someembodiments, the mutein comprises a mutation at L118 that correspond toSEQ ID NO: 6. In some embodiments, the mutation is leucine toisoleucine. In some embodiments, the mutein also comprises a mutation asposition 69, 74, 88, 125, or any combination thereof in these muteinsthat correspond to SEQ ID NO: 6. In some embodiments, the mutation is aV69A mutation. In some embodiments, the mutation is a Q74P mutation. Insome embodiments, the mutation is a N88D or N88R mutation. In someembodiments, the mutation is a C125A or C125S mutation.

In some embodiments, the IL-2 mutein comprises a mutation at one or moreof positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145 thatcorrespond to SEQ ID NO: 15 or one or more positions 29, 31, 35, 37, 48,69, 71, 74, 88, and 125 that correspond to SEQ ID NO: 6. Thesubstitutions can be used alone or in combination with one another. Insome embodiments, the IL-2 mutein comprises substitutions at 2, 3, 4, 5,6, 7, 8, 9, or each of positions 49, 51, 55, 57, 68, 89, 91, 94, 108,and 145. Non-limiting examples such combinations include, but are notlimited to, a mutation at positions 49, 51, 55, 57, 68, 89, 91, 94, 108,and 145; 49, 51, 55, 57, 68, 89, 91, 94, and 108; 49, 51, 55, 57, 68,89, 91, and 94; 49, 51, 55, 57, 68, 89, and 91; 49, 51, 55, 57, 68, and89; 49, 51, 55, 57, and 68; 49, 51, 55, and 57; 49, 51, and 55; 49 and51; 51, 55, 57, 68, 89, 91, 94, 108, and 145; 51, 55, 57, 68, 89, 91,94, and 108; 51, 55, 57, 68, 89, 91, and 94; 51, 55, 57, 68, 89, and 91;51, 55, 57, 68, and 89; 55, 57, and 68; 55 and 57; 55, 57, 68, 89, 91,94, 108, and 145; 55, 57, 68, 89, 91, 94, and 108; 55, 57, 68, 89, 91,and 94; 55, 57, 68, 89, 91, and 94; 55, 57, 68, 89, and 91; 55, 57, 68,and 89; 55, 57, and 68; 55 and 57; 57, 68, 89, 91, 94, 108, and 145; 57,68, 89, 91, 94, and 108; 57, 68, 89, 91, and 94; 57, 68, 89, and 91; 57,68, and 89; 57 and 68; 68, 89, 91, 94, 108, and 145; 68, 89, 91, 94, and108; 68, 89, 91, and 94; 68, 89, and 91; 68 and 89; 89, 91, 94, 108, and145; 89, 91, 94, and 108; 89, 91, and 94; 89 and 91; 91, 94, 108, and145; 91, 94, and 108; 91, and 94; or 94 and 108. Each mutation can becombined with one another. The same substitutions can be made in SEQ IDNO: 6, but the numbering would adjusted appropriately as is clear fromthe present disclosure (20 less than the numbering for SEQ ID NO: 15corresponds to the positions in SEQ ID NO: 6).

In some embodiments, the IL-2 mutein comprises a mutation at one or morepositions of 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO:15 or the equivalent positions at SEQ ID NO: 6 (e.g. positions 15, 16,22, 84, 95, or 126). These mutations can be combined with the otherleucine to isoleucine mutations described herein or the mutation atpositions 73, 76, 100, or 138 that correspond to SEQ ID NO: 15 or at oneor more of positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 6.In some embodiments, the mutation is a E35Q, H36N, Q42E, D104N, E115Q,or Q146E, or any combination thereof. In some embodiments, one or moreof these substitutions is wildtype. In some embodiments, the muteincomprises a wild-type residue at one or more of positions 35, 36, 42,104, 115, or 146 that correspond to SEQ ID NO: 15 or the equivalentpositions at SEQ ID NO: 6 (e.g. positions 15, 16, 22, 84, 95, and 126).

The mutations at these positions can be combined with any of the othermutations described herein, including, but not limited to substitutionsat positions 73, 76, 100, or 138 that correspond to SEQ ID NO: 15 orpositions at one or more of positions 53, 56, 80, or 118 that correspondto SEQ ID NO: 6 described herein and above. In some embodiments, theIL-2 mutein comprises a N49S mutation that corresponds to SEQ ID NO: 15.In some embodiments, the IL-2 mutein comprises a Y51S or a Y51H mutationthat corresponds to SEQ ID NO: 15. In some embodiments, the IL-2 muteincomprises a K55R mutation that corresponds to SEQ ID NO: 15. In someembodiments, the IL-2 mutein comprises a T57A mutation that correspondsto SEQ ID NO: 15. In some embodiments, the IL-2 mutein comprises a K68Emutation that corresponds to SEQ ID NO: 15. In some embodiments, theIL-2 mutein comprises a V89A mutation that corresponds to SEQ ID NO: 15.In some embodiments, the IL-2 mutein comprises a N91R mutation thatcorresponds to SEQ ID NO: 15. In some embodiments, the IL-2 muteincomprises a Q94P mutation that corresponds to SEQ ID NO: 15. In someembodiments, the IL-2 mutein comprises a N108D or a N108R mutation thatcorresponds to SEQ ID NO: 15. In some embodiments, the IL-2 muteincomprises a C145A or C145S mutation that corresponds to SEQ ID NO: 15.These substitutions can be used alone or in combination with oneanother. In some embodiments, the mutein comprises each of thesesubstitutions. In some embodiments, the mutein comprises 1, 2, 3, 4, 5,6, 7, or 8 of these mutations. In some embodiments, the mutein comprisesa wild-type residue at one or more of positions 35, 36, 42, 104, 115, or146 that correspond to SEQ ID NO: 15 or the equivalent positions at SEQID NO: 6 (e.g. positions 15, 16, 22, 84, 95, and 126).

In some embodiments, the IL-2 mutein comprises a N29S mutation thatcorresponds to SEQ ID NO: 6. In some embodiments, the IL-2 muteincomprises a Y31S or a Y31H mutation that corresponds to SEQ ID NO: 6. Insome embodiments, the IL-2 mutein comprises a K35R mutation thatcorresponds to SEQ ID NO: 6. In some embodiments, the IL-2 muteincomprises a T37A mutation that corresponds to SEQ ID NO: 6. In someembodiments, the IL-2 mutein comprises a K48E mutation that correspondsto SEQ ID NO: 6. In some embodiments, the IL-2 mutein comprises a V69Amutation that corresponds to SEQ ID NO: 6. In some embodiments, the IL-2mutein comprises a N71R mutation that corresponds to SEQ ID NO: 6. Insome embodiments, the IL-2 mutein comprises a Q74P mutation thatcorresponds to SEQ ID NO: 6. In some embodiments, the IL-2 muteincomprises a N88D or a N88R mutation that corresponds to SEQ ID NO: 6. Insome embodiments, the IL-2 mutein comprises a C125A or C125S mutationthat corresponds to SEQ ID NO: 6. These substitutions can be used aloneor in combination with one another. In some embodiments, the muteincomprises 1, 2, 3, 4, 5, 6, 7, or 8 of these mutations. In someembodiments, the mutein comprises each of these substitutions. In someembodiments, the mutein comprises a wild-type residue at one or more ofpositions 35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15or the equivalent positions at SEQ ID NO: 6 (e.g. positions 15, 16, 22,84, 95, and 126).

For any of the IL-2 muteins described herein, in some embodiments, oneor more of positions 35, 36, 42, 104, 115, or 146 that correspond to SEQID NO: 15 or the equivalent positions at SEQ ID NO: 6 (e.g. positions15, 16, 22, 84, 95, or 126) are wild-type (e.g. are as shown in SEQ IDNOs: 6 or 15). In some embodiments, 2, 3, 4, 5, 6, or each of positions35, 36, 42, 104, 115, or 146 that correspond to SEQ ID NO: 15 or theequivalent positions at SEQ ID NO: 6 (e.g. positions 15, 16, 22, 84, 95,and 126) are wild-type.

In some embodiments, the IL-2 mutein comprises a sequence of:

(SEQ ID NO: 16) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMC EYADETATIVEFLNRWITFSQSIISTLT

In some embodiments, the IL-2 mutein comprises a sequence of:

(SEQ ID NO: 17) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHIQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMC EYADETATIVEFLNRWITFSQSIISTLT

In some embodiments, the IL-2 mutein comprises a sequence of:

(SEQ ID NO: 18) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHIRPRDLISDINVIVLELKGSETTFMC EYADETATIVEFLNRWITFSQSIISTLT

In some embodiments, the IL-2 mutein comprises a sequence of:

(SEQ ID NO: 19) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMC EYADETATIVEFINRWITFSQSIISTLT

In some embodiments, the IL-2 mutein sequences described herein do notcomprise the IL-2 leader sequence. The IL-2 leader sequence can berepresented by the sequence of MYRMQLLSCIALSLALVTNS (SEQ ID NO: 20).Therefore, in some embodiments, the sequences illustrated above can alsoencompass peptides without the leader sequence. Although SEQ ID NOs;16-20 are illustrated with only mutation at one of positions 73, 76,100, or 138 that correspond to SEQ ID NO: 15 or positions at one or moreof positions 53, 56, 80, or 118 that correspond to SEQ ID NO: 6, thepeptides can comprises one, two, three or 4 of the mutations at thesepositions. In some embodiments, the substitution at each position isisoleucine or other type of conservative amino acid substitution. Insome embodiments, the leucine at the recited positions are substitutedwith, independently, isoleucine, valine, methionine, or phenylalanine.

In some embodiments, the IL-2 mutein molecule is fused to a Fc Region orother linker region as described herein. Examples of such fusionproteins can be found in U.S. Pat. Nos. 9,580,486, 7,105,653, 9,616,105,9,428,567, US2017/0051029, WO2016/164937, US2014/0286898A1,WO2014153111A2, WO2010/085495, WO2016014428A2, WO2016025385A1,US2017/0037102, and US2006/0269515, each of which are incorporated byreference in its entirety.

In some embodiments, the Fc Region comprises what is known as the LALAmutation. Using the Kabat numbering of the Fc region, this wouldcorrespond to L247A, L248A, and G250A. In some embodiments, using the EUnumbering of the Fc region, the Fc region comprises a L234A mutation, aL235A mutation, and/or a G237A mutatoin mutation. Regardless of thenumbering system used, in some embodiments, the Fc portion can comprisemutations that correspond to these residues. In some embodiments, the FcRegion comprises N297G or N297A (kabat numbering) mutations. The Kabatnumbering is based upon a full-length sequence, but would be used in afragment based upon a traditional alignment used by one of skill in theart for the Fc region.

In some embodiments, the Fc Region comprises a sequence of:

(SEQ ID NO: 21) DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG. or(SEQ ID NO: 28) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPG.

In some embodiments, the IL-2 mutein is linked to the Fc Region.Non-limiting examples of linkers are glycine/serine linkers. Forexample, a glycine/serine linkers can be a sequence ofGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22) or GGGGSGGGGSGGGGS (SEQ ID NO: 30).This is simply a non-limiting example and the linker can have varyingnumber of GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29). Insome embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10of the GGGGS (SEQ ID NO: 23) or GGGGA repeats (SEQ ID NO: 29) repeats.

Thus, the IL-2/Fc Fusion can be represented by the formula ofZ_(IL-2M)-L_(gs)-Z_(Fc), wherein Z_(IL-2M) is a IL-2 mutein as describedherein, L_(gs) is a linker sequence as described herein (e.g.glycine/serine linker) and Z_(Fc) is a Fc region described herein orknown to one of skill in the art. In some embodiments, the formula canbe in the reverse orientation Z_(Fc)-L_(gs)-Z_(IL-2M).

In some embodiments, the IL-2/Fc fusion comprises a sequence of

(SEQ ID NO: 24) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATEIKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 25) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHIQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 26) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHIRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPG(SEQ ID NO: 27) MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGISNHKNPRLARMLTFKFYMPEKATELKHLQCLEEELKPLEEALRLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFINRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPG.

In some embodiments, the IL-2/Fc Fusion comprises a sequence selectedfrom the following table, Table 2:

TABLE 2 IL-2/Fc Fusion Protein Amino Acid Sequences SequenceIdentification Sequence SEQ ID NO: 7APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGAGGGGDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLS PGKSEQ ID NO: 8APTSSSTKKTQLQLEHLLLHLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 9APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 10APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSEQ ID NO: 11APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGSEQ ID NO: 12APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNYHTQ KSLSLSPGSEQ ID NO: 13APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 14APTSSSTKKTQLQLEHLLLHLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYPVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPG

In some embodiments, the IL-2 muteins comprises one or more of thesequences provided in the following table, which, in some embodiments,shows the IL-2 mutein fused with other proteins or linkers. The tablealso provides sequences for a variety of Fc domains or variants that theIL-2 can be fused with:

SEQ ID Brief NO: Description Amino Acid Sequence 31 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA with C125STELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE mutationTTFMCEYADETATIVEFLNRWITFSQSIISTLT 32 Human IL-2APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA with C125STELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSE and T3ATTFMCEYADETATIVEFLNRWITFSQSIISTLT mutations 33 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA with N88R andTELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSE C125STTFMCEYADETATIVEFLNRWITFSQSIISTLT 34 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA with V69A,TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISNINVIVLELKGSE Q74P andTTFMCEYADETATIVEFLNRWITFSQSIISTLT C125S mutations 35 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA with V69A,TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE Q74P, N88DTTFMCEYADETATIVEFLNRWITFSQSIISTLT and C125S mutations 36 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA with V69A,TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISRINVIVLELKGSE Q74P, N88RTTFMCEYADETATIVEFLNRWITFSQSIISTLT and C125S mutations 37 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA with N88D andTELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSE C125STTFMCEYADETATIVEFLNRWITFSQSIISTLT 38 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA with L53I,TEIKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE V69A, Q74P,TTFMCEYADETATIVEFLNRWITFSQSIISTLT N88D and C125S mutations 39 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA with L56I,TELKHIQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE V69A, Q74P,TTFMCEYADETATIVEFLNRWITFSQSIISTLT N88D and C125S mutations 40 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA with V69A,TELKHLQCLEEELKPLEEALNLAPSKNFHIRPRDLISDINVIVLELKGSE Q74P, L80I,TTFMCEYADETATIVEFLNRWITFSQSIISTLT N88D and C125S mutations 41 Human IL-2APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA with V69A,TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE Q74P, N88D,TTFMCEYADETATIVEFINRWITFSQSIISTLT L1181, and C125S mutations 42Human IgG1 Fc DKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED(N-terminal PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKfusions) with CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKL234A, L235A, GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGand G237A NVFSCSVMHEALHNHYTQKSLSLSPG mutations 30 GGGGSGGGGSGGGGGGGSGGGGSGGGGS GS linker (15 amino acids) 22 GGGGSGGGGSGGGGGGGSGGGGSGGGGSGGGGS GSGGGGS linker (20 amino acids) 23 GGGGS linkerGGGGS (5 amino acids) 43 Human IgG1 FcDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED (truncated)PEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYK with N297GCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK mutationGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 44 AntibodyASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV Heavy ChainHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEP CH1-CH2-CH3KSCDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVS domainsHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGK (human IgG1EYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC with L234A,LVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW L235A, andQQGNVFSCSVMHEALHNHYTQKSLSLSPG G237A) 45 AntibodyRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG KappaNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK Constant SFNRGECDomain (human) 46 IL-2-G4Sx3-FcAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 47 IL-2 T3A-APASSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA G4Sx3-FcTELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 48 IL-2 N88R-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA G4Sx3-FcTELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISRINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 49 IL-2 V69A,APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA Q74P, -G4Sx3-TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISNINVIVLELKGSE FcTTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 50 IL-2 N88DAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA V69A, Q74P-TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE G4Sx3-FcTTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 51 IL-2 N88RAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA V69A, Q74P-TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISRINVIVLELKGSE G4Sx3-FcTTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 52 IL-2 N88D-APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA G4Sx3-FcTELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 53 IL-2 L53IAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA N88D V69A,TEIKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE Q74P-G4Sx4-FcTTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 54 IL-2 L56IAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA N88D V69A,TELKHIQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE Q74P-G4Sx4-FcTTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 55 IL-2 L801APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA N88D V69A,TELKHLQCLEEELKPLEEALNLAPSKNFHIRPRDLISDINVIVLELKGSE Q74P-G4Sx4-FcTTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 56 IL-2 L118IAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA N88D V69A,TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE Q74P-G4Sx4-FcTTFMCEYADETATIVEFINRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 57 IL-2 N88DAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKA V69A, Q74P-TELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSE G4Sx4-FcTTFMCEYADETATIVEFLNRWITFSQSIISTLTGGGGSGGGGSGGGGSGGGGSDKTHTCPPCPAPEAAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG 58 Fc-G4S-IL-2DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHED N88D V69A,PEVKFNWYVDGVEVHNAKTKPREEQYGSTYRVVSVLTVLHQDWLNGKEYK Q74PCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEALNLAPSKNFHLRPRDLISDINVIVLELKGSETTFMCEYADETATIVEFLN RWITFAQSIISTLT

In some embodiments, the sequences shown in the table or throughoutcomprise or don't comprise one or more mutations that correspond topositions L53, L56, L80, and L118. In some embodiments, the sequencesshown in the table or throughout the present application comprise ordon't comprise one or more mutations that correspond to positions L59I,L63I, I24L, L94I, L96I or L1321 or other substitutions at the samepositions. In some embodiments, the mutation is leucine to isoleucine.In some embodiments, the mutein does not comprise another mutation otherthan as shown or described herein. In some embodiments, the peptidecomprises a sequence of SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 38,SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:43, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47, SEQ IDNO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56, SEQ ID NO:57, or SEQ ID NO: 58.

In some embodiments, the Fc portion of the fusion is not included. Insome embodiments, the peptide consists essentially of a IL-2 muteinprovided for herein. In some embodiments, the protein is free of a Fcportion.

For illustrative purposes only, embodiments of IL-2 mutein fused with aFc and with a targeting moiety are illustrated in FIG. 19.

The sequences are for illustrative purposes only and are not intended tobe limiting. In some embodiments, the compound comprises an amino acidsequence of SEQ ID NO: 53, 54, 55, or 56. In some embodiments, thecompound comprises an amino acid sequence of SEQ ID NO: 53, 54, 55, or56.with or without a C125A or C125S mutation.

In an embodiment, an IL-2 mutein molecule comprises at least 60, 70, 80,85, 90, 95, or 97% sequence identity or homology with a naturallyoccurring human IL-2 molecule, e.g., a naturally occurring IL-2 sequencedisclosed herein or those that incorporated by reference.

As described herein the IL-2 muteins can be part of a bi-specificmolecule with a tethering moiety, such as a MAdCAM antibody that willtarget the IL-2 mutein to a MAdCAM expressing cell. As described herein,the bispecific molecule can be produced from two polypeptide chains. Insome embodiments, the following can be used:

TABLE OF MADCAM-IL-2 MUTEIN BISPECIFIC COMPOUNDS Chain 2 N-terminalto C-terminal Chain 1 N-terminal to C-terminal MoleculeMolecule Component Component Sequence IDs Sequence IDs Antibody HeavyChain Light CH1-CH2- C- Chain Antibody VH CH3 terminal Light Chain CKDetail Domain Domains Linker 1 Moiety VK Domain Domain 1. Anti-Rat Anti- SEQ ID SEQ ID SEQ ID Rat Anti- SEQ MAdCam-Fc- MAdCam -VH1NO: 44 NO: 23 NO: 35 MAdCam -VK1 ID IL-2 N88D NO: V69A, Q74P 45 2. Anti-Rat Anti- SEQ ID SEQ ID SEQ ID Rat Anti- SEQ MAdCam - MAdCam-VH2 NO: 44NO: 23 NO: 35 MAdCam -VK2 ID Fc-IL-2 NO: N88D V69A, 45 Q74P 3. Anti-Rat Anti- SEQ ID SEQ ID SEQ ID Rat Anti- SEQ MAdCam - MAdCam -VH1 NO: 44NO: 23 NO: 41 MAdCam -VK3 ID Fc-IL-2 NO: L118I N88D 45 V69A, Q74P4. TTJ2- Human TTJ2- SEQ ID SEQ ID SEQ ID Human TTJ2- SEQ Fc-IL-2 VHNO: 44 NO: 23 NO: 41 VK ID L118I N88D NO: V69A, Q74P 45 5. antiAnti-MAdCam SEQ ID SEQ ID SEQ ID Anti-MAdCam SEQ hu.MAdCAM- Human VH3NO: 44 NO: 23 NO: 41 Human VK3 ID Fc-IL-2 NO: L118I N88D 45 V69A, Q74P6. anti Anti-MAdCam SEQ ID SEQ ID SEQ ID Anti-MAdCam SEQ hu.MAdCAM-Human VH4 NO: 44 NO: 23 NO: 41 Human VK4 ID Fc-IL-2 NO: L118I N88D 45V69A, Q74P 7. anti Anti-MAdCam SEQ ID SEQ ID SEQ ID Anti-MAdCam SEQhu.MAdCAM- Human VHS NO: 44 NO: 23 NO: 41 Human VK5 ID Fc-IL-2 NO:L118I N88D 45 V69A, Q74P

The proteins can be produced with or without a C125A or C125S mutationin the IL-2 mutein.

In some embodiments, the constant kappa domain in any of the lightchains can be replaced with a constant lambda domain.

GITR-Binders

GITR(CD357) is a cell surface marker present on Tregs. Blockade of theGITR-GITRL interaction maintains Treg function. In some embodiments, atherapeutic compound comprises an IIC binding entity that bindsGITR-expressing Treg cells and a targeting moiety that targets thetherapeutic compound to the target tissue of interest.

In some embodiments, a therapeutic compound comprises an anti-GITRantibody molecule, e.g., anti-GITR antibody molecule that inhibitbinding of GITR to GITRL.

In some embodiments, a therapeutic compound comprises an anti-GITRantibody molecule, anti-GITR antibody molecule that inhibit binding ofGITR to GITRL, and PD-1 agonist, IL-2 mutein molecule, or other effectordescribed herein.

While not wishing to be bound by theory, it is believed that thetherapeutic compound that comprises a GITR binder effects accumulationof GITR-expressing Tregs at the site targeted by the targeting moiety ofthe therapeutic compound, e.g., a transplant or site of organ injury.

Butyrophilins/Butyrophilin-Like Molecules

Effector binding/modulating moiety can comprise an agonistic BTNL2molecule. While not wishing to be bound by theory it is believed thatagonistic BTNL2 molecules induce Treg cells.

An agonistic BTNL2 molecule as that term as used herein, refers to apolypeptide having sufficient BTNL2 sequence that, as part of atherapeutic compound, it induces Treg cells. In some embodiments, aBTNL2 molecule has at least 60, 70, 80, 90, 95, 99, or 100% sequenceidentity, or substantial sequence identity, with a naturally occurringbutyrophilin.

In some embodiments, an effector binding/modulating moiety anantagonistic BTNL8 molecule.

Therapeutic Compounds Comprising an SM Binding/Modulating Moiety:Manipulation of Local Microenvironment

A therapeutic compound can comprise an effector binding/modulatingmoiety that promotes an immuno-suppressive local microenvironment, e.g.,by providing in the proximity of the target, a substance that inhibitsor minimizes attack by the immune system of the target, referred toherein a SM binding/modulating moiety.

In some embodiments, the SM binding/modulating moiety comprises amolecule that inhibits or minimizes attack by the immune system of thetarget (referred to herein as an SM binding/modulating moiety). In someembodiments, a therapeutic compound comprises an SM binding/modulatingmoiety that binds and accumulates a soluble substance, e.g., anendogenous or exogenous substance having immunosuppressive function. Insome embodiments, a therapeutic compound comprises an SMbinding/modulating moiety, e.g., a CD39 molecule or a CD73 molecule oralkaline phosphatase molecule, that binds, inhibits, sequesters,degrades or otherwise neutralizes a soluble substance, typically andendogenous soluble substance, e.g., ATP in the case of a CD39 moleculeor alkaline phosphatase molecule, or AMP in the case of a CD73 molecule,that promotes immune attack. In some embodiments, a therapeutic compoundcomprises an SM binding/modulating moiety that comprises animmune-suppressive substance, e.g. a fragment of protein that isimmunosuppressive.

Therapeutic Compounds Comprising an ICSM Binding/Modulating Moiety:Inhibition of Stimulation, e.g., Inhibition of Co-Stimulation of ImmuneCells

A therapeutic compound can comprise an ICSM binding/modulating moietythat inhibits or antagonizes a stimulatory, e.g., co-stimulatory bindingpair, e.g., OX40 and OX40L. The ICSM binding/modulating moiety can bindand antagonize either member of the pair.

In an embodiment, the ICSM binding/modulating moiety comprises anantibody molecule that binds and antagonizes either member of astimulatory, e.g., co-stimulatory binding pair. In an embodiment theICSM binding/modulating moiety comprises antagonistic analog of one ofthe members of the binding pair. In such embodiments the ICSMbinding/modulating moiety can comprise a soluble fragment of one of themembers that binds the other. Typically the analog will have at least50, 60, 70, 80, 90, 95, or 98% homology or sequence identity with anaturally occurring member that binds the target member of the pair. Inthe case of an ICSM binding/modulating moiety that binds the memberpresent on the surface of an immune cell, the ICSM binding/modulatingmoiety typically binds but does not activate, or allow endogenouscounter member to bind and activate.

Thus, in the case of the binding pair that includes, for example, theOX40 immune cell member and the OX40L counter member, an ICSMbinding/modulating member can comprise any of the following:

a) an antibody molecule that binds the OX40 immune cell member andantagonizes stimulation, e.g., by blocking binding of endogenous OX40Lcounter member;

b) an antibody molecule that binds OX40L counter member and antagonizesstimulation, e.g., by blocking effective binding of the endogenous OX40Lcounter member to the OX40 immune cell member;

c) a soluble fragment or analog of OX40L counter member which binds OX40immune cell member and antagonizes stimulation; and

c) a soluble fragment or analog of OX40 immune cell member which bindsOX40L counter member and antagonizes stimulation.

For example, the ICSM binding/modulating moiety, e.g., an antibodymolecule or an antagonistic analog or of the counter member, can bind toCD2, ICOS, CD40L, CD28, LFA1, SLAM, TIM1, CD30, OX40 (CD134), 41BB(CD137), CD27, HVEM, DR3, GITR, BAFFR, TACI, BCMA, or CD30, CD40. Inanother embodiment, the ICSM binding/modulating moiety, e.g., anantibody molecule or an antagonistic analog or of the counter member,can bind to B7.1, B7.2, ICOSL (B7-H2, B7RP1), LFA3, CD48, CD58, ICAM1,SLAM, TIM4, CD40, CD30L, OX40L (CD252), 41BBL (CD137L), CD70, LIGHT,TL1A, GITRL, BAFF, APRIL, or CD30, CD40L.

In some embodiments, the ICSM binding/modulating molecule binds, andantagonizes, an activating or costimulatory molecule, e.g., acostimulatory molecule, present on an immune cell, or binds the countermember preventing the counter member from activating the costimulatorymolecule present on the immune cell. In some embodiments, the ICSMcomprises an antagonistic antibody molecule e.g., an antibody moleculethat binds the costimulatory molecule on an immune cell or binds thecounter member of the ICSM, preventing the counter member fromactivating the costimulatory molecule on the immune cell, and results ininhibiting the activity of the costimulatory molecule. In someembodiments, the ICSM comprises an antagonistic counterpart molecule,e.g., a fragment of a molecule that binds the costimulatory molecule,and results in the inhibition of the costimulatory molecule activity.

In some embodiments, one member of the binding pair will be on thesurface of an immune cell, e.g., a T, B, or NK cell or dendritic cell,while the counter member will be on another immune cell, or an APC suchas a dendritic cell or on non-immune cells such as smooth cells, orendothelial cells.

The following table provides non-limiting examples of costimulatorymolecule and counterstructure pairs

TABLE 2 Costimulatory molecule and counterstructure pairs CostimulatoryMolecule (eg on T cells) Counterstructure CD28 B7.1 or B7.2 ICOS ICOSL(B7H-2, B7RP1) CD2 LFA3, CD48, CD58 LFA1 ICAM1 SLAM SLAM TIM1 TIM4 CD40LCD40 CD30 CD30L OX40/CD134 OX40L (CD252) 41BB/CD137 41BBL (CD137L) CD27CD70 HVEM LIGHT DR3 TL1A GITR GITRL Costimulatory Molecule (eg on Bcells) Counterstructure BAFFR BAFF TACI BAFF and APRIL BCMA BAFF andAPRIL CD40 CD40L CD30L CD30Donor Tissue

Therapeutic compounds and methods described herein can be used inconjunction with a transplantation of donor tissue into a subject andcan minimizes rejection of, minimizes immune effector cell mediateddamage to, prolongs acceptance of, or prolongs the functional life of,donor transplant tissue. The tissue can be xenograft or allografttissue. Transplanted tissue can comprise all or part of an organ, e.g.,a liver, kidney, heart, pancreas, thymus, skin or lung. In embodiments,therapeutic compounds described herein reduce, or eliminate the need forsystemic immune suppression. Therapeutic compounds and methods describedherein can also be used to treat GVHD. In some embodiments, host cellsare coated with a therapeutic compound that comprises, as an effectorbinding/modulating moiety, a PD-L1 molecule.

Table 2 provides target molecules for transplant indications. A targetmolecule is the target to which a targeting moiety binds. As discussedelsewhere herein, In some embodiments, a targeting moiety is selectedthat binds a product of an allele present on donor tissue and which isnot expressed by the subject (recipient) or at expressed at a differentlevel (e.g. reduced or substantially reduced).

TABLE 2 Target Molecules for Transplant Indications IndicationOrgan/cell type Target Allograft transplant tissue, e.g., All HLA-A,HLA-B, HLA-C, allograft solid organ transplant, HLA-DP, HLA-DQ or HLA-DRGyHD Transplant Kidney Antigens expressed in the kidney where immunecells infiltrate, for example including but not limited to the tubularinterstitial region eg Uromodulin, SLC22A2, SLC22A6, FXYD4, SLC5A10,SLC6A13, AQP6, SLC13A3, TMEM72, BSND, NPR3, and the proximal and distaltubular epithelium, such as OAT1, OCT2Auto-Immune Disorders

Therapeutic compounds and methods described herein can be used to treata subject having or at risk for having an unwanted autoimmune response,e.g., an auto immune response in Type 1 Diabetes, Multiple Sclerosis,Cardiomyositis, vitiligo, alopecia, inflammatory bowel disease (IBD,e.g. Crohn's disease or ulcerative colitis), Sjogren's syndrome, focalsegmented glomerular sclerosis (FSGS), scleroderma/systemic sclerosis(SSc) or rheumatoid arthritis. In some embodiments, the treatmentminimizes rejection of, minimizes immune effector cell mediated damageto, prolongs the survival of subject tissue undergoing, or a risk for,autoimmune attack. Table 3 provides target molecules for severalautoimmune indications and organ/cell types. A target molecule is thetarget to which a targeting moiety binds.

TABLE 3 Target Molecules for autoimmune indications IndicationOrgan/cell type Target Molecule Type 1 Diabetes and Pancreas/Pancreaticislets, SEZ6L2, LRP11, DISP2, Transplant beta cells SLC30A8, FXYD2TSPAN7 TMEM27 (reference Hald et al. 2012 Diabetelogia 55:154); FXYD2;GPR119; HEPACAM2, DPP6, or MAdCAM Multiple Sclerosis CNS/myelin sheathof MOG, PLP, MBP oligodendrocytes Cardiomyositis, rheumatoidCardiomyocytes, monocytes, SIRPA (CD172a) arthritis macrophages, myeloidcells Inflammatory bowel disease Intestine MAdCAM (ulcerative colitis,Crohn's disease) or GVHD; Celiac disease Autoimmune hepatitis (AIH);liver MAdCAM Primary Sclerosing Cholangitis (PSC); Primary BiliarySclerosis; (PBC); transplant Focal Segmented Glomerular Kidney,podocytes, tubules, COL1A1, Cadherin 2, Sclerosis (FSGS) and otherepithelial cells VCAM-1, Thyl, Podocin, diseases that can affect kidneyKIM1 (Hodgin et al, Am J for example lupus nephritis, Pathol 177:16752010); systemic scleroderma, PLA2R; OAT1; OCT2; K- membranous glomerularcadherin 6 nephropathy (MGN); Membranous nephropathy (MN); MinimalChange Disease (MCD); IgA nephropathy; ANCA- associated vasculitis (AAV)Sjogren's syndrome Salivary glands, epithelial FCGR3B, HLAB, KIM1 (Hucells, kidney et al Arth and Rheum 56:3588 2007 Scleroderma, systemicskin, kidney, lung, Fibroblasts, Collagen I, III, VI, VII, sclerosis(SSc) connective tissue fibronectin (Wang et al Arth and Rheum 54:22712006) vitiligo Skin, epidermis, Langerhans COL17A1, CD1A, CD207, cells,keratinocytes, desmoglein 1-4, keratin 1 melanocytes Alopecia areataSkin, Hair follicle/hair bulb, CD133 (Yang and Cotsarelis, dermis JDermatol Sci 57:2 2010)

Other examples of autoimmune disorders and diseases that can be treatedwith the compounds described herein include, but are not limited to,Myocarditis, Postmyocardial infarction syndrome, Postpericardiotomysyndrome, Subacute bacterial endocarditis, Anti-Glomerular BasementMembrane nephritis, Interstitial cystitis, Lupus nephritis, membranousglomerulonephropathy, Chronic Kidney Disease (“CKD”), Autoimmunehepatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis,Antisynthetase syndrome, alopecia areata, autoimmune angioedema,autoimmune progesterone dermatitis, autoimmune urticaria, bullouspemphigoid, cicatricial pemphigoid, dermatitis herpetiformis, discoidlupus erythematosus, epidermolysis bullosa acquisita, erythema nodosum,gestational pemphigoid, hidradenitis suppurativa, lichen planus, lichensclerosus, linear iga disease (lad), morphea, pemphigus vulgaris,pityriasis lichenoides et varioliformis acuta, mucha-habermann disease,psoriasis, systemic scleroderma, vitiligo, Addison's disease, Autoimmunepolyendocrine syndrome (APS) type 1, Autoimmune polyendocrine syndrome(APS) type 2, Autoimmune polyendocrine syndrome (APS) type 3, Autoimmunepancreatitis (AIP), Diabetes mellitus type 1, Autoimmune thyroiditis,Ord's thyroiditis, Graves' disease, Autoimmune Oophoritis,Endometriosis, Autoimmune orchitis, Sjogren's syndrome, Autoimmuneenteropathy, Coeliac disease, Crohn's disease, Microscopic colitis,Ulcerative colitis, thrombocytopenia, Adiposis, dolorosa, Adult-onsetStill's, disease, Ankylosing, Spondylitis, CREST syndrome, Drug-inducedlupus, Enthesitis-related arthritis, Eosinophilic fasciitis, Feltysyndrome, IgG4-related disease, Juvenile, Arthritis, Lyme disease(Chronic), Mixed connective tissue disease (MCTD), Palindromicrheumatism, Parry Romberg syndrome, Parsonage-Turner syndrome, Psoriaticarthritis, Reactive arthritis, Relapsing polychondritis, Retroperitonealfibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schnitzlersyndrome, Systemic Lupus Erythematosus (SLE), Undifferentiatedconnective tissue disease (UCTD), Dermatomyositis, Fibromyalgia,Inclusion body myositis, Myositis, Myasthenia gravis, Neuromyotonia,Paraneoplastic cerebellar degeneration, Polymyositis, Acute disseminatedencephalomyelitis (ADEM), Acute motor axonal neuropathy,Anti-N-Methyl-D-Aspartate (anti-NMDA) Receptor Encephalitis, Baloconcentric sclerosis, Bickerstaff's encephalitis, Chronic inflammatorydemyelinating polyneuropathy, Guillain-Barre syndrome, Hashimoto'sencephalopathy, Idiopathic inflammatory demyelinating diseases,Lambert-Eaton myasthenic syndrome, Multiple sclerosis, Oshtoransyndrome, Pediatric Autoimmune Neuropsychiatric Disorder Associated withStreptococcus (PANDAS), Progressive inflammatory neuropathy, Restlessleg syndrome, Stiff person syndrome, Sydenham chorea, Transversemyelitis, Autoimmune retinopathy, Autoimmune uveitis, Cogan syndrome,Graves ophthalmopathy, Intermediate uveitis, Ligneous conjunctivitis,Mooren's ulcer, Neuromyelitis optica, Opsoclonus myoclonus syndrome,Optic neuritis, Scleritis, Susac's syndrome, Sympathetic ophthalmia,Tolosa-Hunt syndrome, Autoimmune inner ear disease (AIED), Meniere'sdisease, Behcet's disease, Eosinophilic granulomatosis with polyangiitis(EGPA), Giant cell arteritis, Granulmatosis with polyangiitis (GPA), IgAvasculitis (IgAV), Kawasaki's disease, Leukocytoclastic vasculitis,Lupus vasculitis, Rheumatoid vasculitis, Microscopic polyangiitis (MPA),Polyarteritis nodosa (PAN), Polymyalgia rheumaticia, Vasculitis, PrimaryImmune Deficiency, and the like.

Other examples of potential autoimmune disorders and diseases, as wellas autoimmune comorbidities that can be treated with the compoundsdescribed herein include, but are not limited to, Chronic fatiguesyndrome, Complex regional pain syndrome, Eosinophilic esophagitis,Gastirtis, Interstitial lung disease, POEMS syndrome, Raynaud'sphenomenon, Primary immunodeficiency, Pyoderma gangrenosum,Agammaglobulinemia, Anyloidosis, Anyotrophic lateral sclerosis,Anti-tubular basement membrane nephritis, Atopic allergy, Atopicdermatitis, Autoimmune peripheral neuropathy, Blau syndrome, Castleman'sdisease, Chagas disease, Chronic obstructive pulmonary disease, Chronicrecurrent multifocal osteomyelitis, Complement component 2 deficiency,Contact dermatitis, Cushing's syndrome, Cutaneous leukocytoclasticangiitis, Dego' disease, Eczema, Eosinophilic gastroenteritis,Eosinophilic pneumonia, Erythroblastosis fetalsis, Fibrodysplasiaossificans progressive, Gastrointestinal pemphigoid,Hypogammaglobulinemia, Idiopathic giant-cell myocarditis, Idiopathicpulmonary fibrosis, IgA nephropathy, Immunregulatory lipoproteins, IPEXsyndrome, Ligenous conjunctivitis, Majeed syndrome, Narcolepsy,Rasmussen's encephalitis, Schizophrenia, Serum sickness,Spondyloathropathy, Sweet's syndrome, Takayasu's arteritis, and thelike.

In some embodiments, the autoimmune disorder does not comprise pemphigusVulgaris, pemphigus. In some embodiments, the autoimmune disorder doesnot comprise pemphigus foliaceus. In some embodiments, the autoimmunedisorder does not comprise bullous pemphigoid. In some embodiments, theautoimmune disorder does not comprise Goodpasture's Disease. In someembodiments, the autoimmune disorder does not comprise psoriasis. Insome embodiments, the autoimmune disorder does not comprise a skindisorder. In some embodiments, the disorder does not comprise aneoplastic disorder, e.g., cancer.

Therapeutic Compounds

A therapeutic compound comprises a specific targeting moietyfunctionally associated with an effector binding/modulating moiety. Insome embodiments, the specific targeting moiety and effectorbinding/modulating moiety are linked to one another by a covalent ornoncovalent bond, e.g., a covalent or non-covalent bond directly linkingthe one to the other. In other embodiments, a specific targeting moietyand effector binding/modulating moiety are linked, e.g., covalently ornoncovalently, through a linker moiety. E.g., in the case of a fusionpolypeptide, a polypeptide sequence comprising the specific targetingmoiety and a polypeptide sequence can be directly linked to one anotheror linked through one or more linker sequences. In some embodiments, thelinker moiety comprises a polypeptide. Linkers are not, however, limitedto polypeptides. In some embodiments, a linker moiety comprises otherbackbones, e.g., a non-peptide polymer, e.g., a PEG polymer. In someembodiments, a linker moiety can comprise a particle, e.g., ananoparticle, e.g., a polymeric nanoparticle. In some embodiments, alinker moiety can comprise a branched molecule, or a dendrimer. However,in embodiments where the effector binding/modulating moiety comprises anICIM binding/modulating moiety (which binds an effector like PD-1)structures that result in clustering in the absence of target bindingshould be avoided as they may cause clustering in the absence of targetbinding. Thus in embodiments, the therapeutic compound has a structure,e.g., the copies of an ICIM are sufficiently limited, such thatclustering in the absence of target binding is minimized orsubstantially eliminated, or eliminated, or is sufficiently minimizedthat substantial systemic immune suppression does not occur.

In some embodiments, a therapeutic compound comprises a polypeptidecomprising a specific targeting moiety covalently or non-covalentlyconjugated to an effector binding/modulating moiety. In someembodiments, a therapeutic molecule comprises a fusion protein havingcomprising a specific targeting moiety fused, e.g., directly or througha linking moiety comprising one or more amino acid residues, to aneffector binding/modulating moiety. In some embodiments, a therapeuticmolecule comprises a polypeptide comprising a specific targeting moietylinked by a non-covalent bond or a covalent bond, e.g., a covalent bondother than a peptide bond, e.g., a sulfhydryl bond, to an effectorbinding/modulating moiety.

In some embodiments, a therapeutic compound comprises polypeptide, e.g.,a fusion polypeptide, comprising:

1.a) a specific targeting moiety comprising a target specific bindingpolypeptide;

1.b) a specific targeting moiety comprising a target ligand bindingmolecule;

1.c) a specific targeting moiety comprising an antibody molecule;

1.d) a specific targeting moiety comprising a single chain antibodymolecule, e.g., a scFv domain; or

1.e) a specific targeting moiety comprising a first of the light orheavy chain variable region of an antibody molecule, and wherein theother variable region is covalently or non covalently associated withthe first;

and

2.a) an effector binding/modulating moiety comprising an effectorspecific binding polypeptide;

2.b) an effector binding/modulating moiety comprising an effector ligandbinding molecule;

2.c) an effector binding/modulating moiety comprising an antibodymolecule;

2.d) an effector binding/modulating moiety comprising a single chainantibody molecule, e.g., a scFv domain; or

2.e) an effector binding/modulating moiety comprising a first of thelight or heavy chain variable region of an antibody molecule, andwherein the other variable region is covalently or non covalentlyassociated with the first.

In some embodiments, a therapeutic compound comprises 1.a and 2.a.

In some embodiments, a therapeutic compound comprises 1.a and 2.b.

In some embodiments, a therapeutic compound comprises 1.a and 2.c.

In some embodiments, a therapeutic compound comprises 1.a and 2.d.

In some embodiments, a therapeutic compound comprises 1.a and 2.e.

In some embodiments, a therapeutic compound comprises 1.b and 2.a.

In some embodiments, a therapeutic compound comprises 1.b and 2.b.

In some embodiments, a therapeutic compound comprises 1.b and 2.c.

In some embodiments, a therapeutic compound comprises 1.b and 2.d.

In some embodiments, a therapeutic compound comprises 1.b and 2.e.

In some embodiments, a therapeutic compound comprises 1.c and 2.a.

In some embodiments, a therapeutic compound comprises 1.c and 2.b.

In some embodiments, a therapeutic compound comprises 1.c and 2.c.

In some embodiments, a therapeutic compound comprises 1.c and 2.d.

In some embodiments, a therapeutic compound comprises 1.c and 2.e.

In some embodiments, a therapeutic compound comprises 1.d and 2.a.

In some embodiments, a therapeutic compound comprises 1.d and 2.b.

In some embodiments, a therapeutic compound comprises 1.d and 2.c.

In some embodiments, a therapeutic compound comprises 1.d and 2.d.

In some embodiments, a therapeutic compound comprises 1.d and 2.e.

In some embodiments, a therapeutic compound comprises 1.e and 2.a.

In some embodiments, a therapeutic compound comprises 1.e and 2.b.

In some embodiments, a therapeutic compound comprises 1.e and 2.c.

In some embodiments, a therapeutic compound comprises 1.e and 2.d.

In some embodiments, a therapeutic compound comprises 1.e and 2.e.

Therapeutic compounds disclosed herein can, for example, comprise aplurality of effector binding/modulating and specific targetingmoieties. Any suitable linker or platform can be used to present theplurality of moieties. The linker is typically coupled or fused to oneor more effector binding/modulating and targeting moieties.

In some embodiments, two (or more) linkers associate, either covalentlyor noncovalently, e.g., to form a hetero or homo-dimeric therapeuticcompound. E.g., the linker can comprise an Fc region and two Fc regionsassociate with one another. In some embodiments of a therapeuticcompound comprising two linker regions, the linker regions can selfassociate, e.g., as two identical Fc regions. In some embodiments of atherapeutic compound comprising two linker regions, the linker regionsare not capable of, or not capable of substantial, self association,e.g., the two Fc regions can be members of a knob and hole pair.

Non-limiting exemplary configurations of therapeutic compounds comprisethe following (e.g., in N to C terminal order):

R1—Linker Region A—R2

R3—Linker Region B—R4,

-   wherein,

R1, R2, R3, and R4, each independently comprises an effectorbinding/modulating moiety, e.g., an ICIM binding/modulating moiety, anIIC binding/modulating moiety, ICSM binding/modulating moiety, or an SMbinding/modulating moiety; a specific targeting moiety; or is absent;

Linker Region A and Linker B comprise moieties that can associate withone another, e.g., Linker A and Linker B each comprises an Fc moietyprovided that an effector binding/modulating moiety and a specifictargeting moiety are present.

In some embodiments:

R1 comprises an effector binding/modulating moiety, e.g., an ICIMbinding/modulating moiety, an IIC binding/modulating moiety, ICSMbinding/modulating moiety, or an SM binding/modulating moiety, or isabsent;

R2 comprises a specific targeting moiety, or is absent;

R3 comprises an effector binding/modulating moiety, e.g., an ICIMbinding/modulating moiety, an IIC binding/modulating moiety, ICSMbinding/modulating moiety, or an SM binding/modulating moiety, or isabsent;

R4 comprises a specific targeting moiety, or is absent;

Linker Region A and Linker B comprise moieties that can associate withone another, e.g., Linker A and Linker B each comprises an Fc moiety,provided that one of R1 or R3 is present and one of R2 or R4 is present.

In some embodiments:

R1 comprises a specific targeting moiety, or is absent;

R2 comprises an effector binding/modulating moiety, e.g., an ICIMbinding/modulating moiety, an IIC binding/modulating moiety, ICSMbinding/modulating moiety, or an SM binding/modulating moiety, or isabsent;

R3 comprises a specific targeting moiety, or is absent;

R4 comprises an effector binding/modulating moiety, e.g., an ICIMbinding/modulating moiety, an IIC binding/modulating moiety, ICSMbinding/modulating moiety, or an SM binding/modulating moiety, or isabsent;

Linker Region A and Linker B comprise moieties that can associate withone another, e.g., Linker A and Linker B each comprises an Fc moiety,provided that one of R1 or R3 is present and one of R2 or R4 is present.

-   Non-limiting examples include, but are not limited to:

Linker Linker R1 Region A R2 R3 Region B R4 Other HCVR and Fc RegionfcFv HCVR and Fc Region scFv Self Pairing LCVR LCVR Linker Regions HCVRand Fc Region fcFv HCVR and Fc Region scFv Non-Self LCVR LCVR Pairinglinker regions HCVR and Fc Region fcFv HCVR and Fc Region scFv SelfPairing LCVR (or LCVR (or Linker Regions absent) absent) One of R1 or R3is absent. HCVR and Fc Region fcFv HCVR and Fc Region scFv Non-Self LCVR(or LCVR (or Pairing Linker absent) absent) Regions One of R1 or R3 isabsent. HCVR and Fc Region fcFv (or HCVR and Fc Region scFv (or SelfPairing LCVR absent) LCVR absent) linker regions One of R2 or R4 isabsent. HCVR and Fc Region fcFv (or HCVR and Fc Region scFv (or Non-SelfLCVR absent) LCVR absent) Pairing linker regions One of R2 or R4 isabsent. HCVR and Fc Region fcFv HCVR and Fc Region scFv Self PairingLCVR LCVR Linker Regions R1 and R3 are the same HCVR and Fc Region fcFvHCVR and Fc Region scFv Non-Self LCVR LCVR Pairing linker regions R1 andR3 are different HCVR and Fc Region fcFv HCVR and Fc Region scFv SelfPairing LCVR LCVR Linker Regions R2 and R4 are the same HCVR and FcRegion fcFv HCVR and Fc Region scFv Non-Self LCVR LCVR Pairing linkerregions R2 and R4 are different HCVR and LCVR: refers to an moietycomprising an antigen binding portion of a heavy and light chianvariable region, typically with the heavy chain fused to the Linkerregion. Self pairing: wherein a liker region can pair with itself, e.g.,an Fc region that can pair a copy of itself. Non-Self Pairing: wherein aLinker Region does not pair with itself, or does not substantially pairwith itself, e.g., an Fc region does not or does not significantly pairwith itself, e.g., wherein Linker Region A and Linker Region B aremembers of a knob and hole pair.

In some embodiments:

R1, R2, R3 and R4 each independently comprise: an effector bindingmodulating moiety that activates an inhibitory receptor on an immunecell, e.g., a T cell or a B cell, e.g., a PD-L1 molecule or a functionalanti-PD-1 antibody molecule (an agonist of PD-1); a specific targetingmoiety; or is absent;

provided that an effector binding moiety and a specific targeting moietyare present.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties).

In some embodiments:

R1 and R3 independently comprise an effector binding modulating moietythat activates an inhibitory receptor on an immune cell, e.g., a T cellor a B cell, e.g., a PD-L1 molecule or an functional anti-PD-1 antibodymolecule (an agonist of PD-1); and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties).

In some embodiments:

R1 and R3 independently comprise a functional anti-PD-1 antibodymolecule (an agonist of PD-1); and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties).

In some embodiments:

R1 and R3 independently comprise specific targeting moieties, e.g., ananti-tissue antigen antibody; and

R2 and R4 independently comprise a functional anti-PD-1 antibodymolecule (an agonist of PD-1), e.g., an scFv molecule.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties).

In some embodiments:

R1 and R3 independently comprise a PD-L1 molecule (an agonist of PD-1);and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen; and

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties).

In some embodiments:

R1 and R3 independently comprise specific targeting moieties, e.g., ananti-tissue antigen antibody; and

R2 and R4 independently comprise a PD-L1 molecule (an agonist of PD-1).

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties).

In some embodiments:

R1, R2, R3 and R4 each independently comprise: an SM binding/modulatingmoiety which modulates, e.g., binds and inhibits, sequesters, degradesor otherwise neutralizes a substance, e.g., a soluble molecule thatmodulates an immune response, e.g., ATP or AMP, e.g., a CD39 molecule ora CD73 molecule; a specific targeting moiety; or is absent;

provided that an SM binding/modulating moiety and a specific targetingmoiety are present.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

R1 and R3 independently comprise an SM binding/modulating moiety whichmodulates, e.g., binds and inhibits, sequesters, degrades or otherwiseneutralizes a substance, e.g., a soluble molecule that modulates animmune response, e.g., ATP or AMP, e.g., a CD39 molecule or a CD73molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

R1 and R3 independently comprise a CD39 molecule or a CD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

R1 and R3 each comprises a CD39 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen; and

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

R1 and R3 each comprises a CD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

One of R1 and R3 comprises a CD39 molecule and the other comprises aCD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

R1, R2, R3 and R4 each independently comprise: an HLA-G molecule; aspecific targeting moiety; or is absent;

provided that an HLA-G molecule and a specific targeting moiety arepresent.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

R1 and R3 each comprise an HLG-A molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

R1 and R3 each comprise an agonistic anti-LILRB1 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

R1 and R3 each comprise an agonistic anti-KIR2DL4 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

R1 and R3 each comprise an agonistic anti-LILRB2 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

R1 and R3 each comprise an agonistic anti-NKG2A antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

one of R1 and R3 comprises a first moiety chosen from, and the othercomprises a different moiety chosen from: an antagonistic anti-LILRB1antibody molecule, an agonistic anti-KR2DL4 antibody molecule, and anagonistic anti-NKG2A antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

one of R1 and R3 comprises an antagonistic anti-LILRB1 antibody moleculeand the other comprises an agonistic anti-KR2DL4 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments:

one of R1 and R3 comprises an antagonistic anti-LILRB1 antibody moleculeand the other comprises an agonistic anti-NKG2A antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In an embodiment:

R1, R2, R3 and R4 each independently comprise: an IL-2 mutein molecule;a specific targeting moiety; or is absent;

provided that an IL-2 mutein molecule and a specific targeting moietyare present.

In an embodiment Linker A and Linker B comprise Fc moieties (e.g., selfpairing Fc moieties or Fc moieties that do not, or do not substantiallyself pair).

One of R1, R2, R3 and R4 comprises an IL-2 mutein molecule, onecomprises an anti-GITR antibody molecule, e.g., an anti-GITR antibodymolecule that inhibits binding of GITRL to GITR, and one comprises aspecific targeting moiety;

In an embodiment Linker A and Linker B comprise Fc moieties (e.g., selfpairing Fc moieties or Fc moieties that do not, or do not substantiallyself pair).

In an embodiment:

R1 and R3 each comprise an IL-2 mutein molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In an embodiment Linker A and Linker B comprise Fc moieties (e.g., selfpairing Fc moieties or Fc moieties that do not, or do not substantiallyself pair).

In an embodiment:

one of R1 and R3 comprises a GARP binding molecule, e.g., an anti-GARPantibody molecule or a GITR binding molecule, e.g., an anti-GITRantibody molecule and the other comprises an IL-2 mutein molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In an embodiment Linker A and Linker B comprise Fc moieties (e.g., selfpairing Fc moieties or Fc moieties that do not, or do not substantiallyself pair).

In an embodiment:

one of R1 and R3 comprises a GARP binding molecule, e.g., an anti-GARPantibody molecule and the other comprises an IL-2 mutein molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In an embodiment Linker A and Linker B comprise Fc moieties (e.g., selfpairing Fc moieties or Fc moieties that do not, or do not substantiallyself pair).

In an embodiment:

one of R1 and R3 comprises a GITR binding molecule, e.g., an anti-GITRantibody molecule, and the other comprises an IL-2 mutein molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In an embodiment Linker A and Linker B comprise Fc moieties (e.g., selfpairing Fc moieties or Fc moieties that do not, or do not substantiallyself pair).

In some embodiments:

R1, R2, R3 and R4 each independently comprise: an effector bindingmodulating moiety that activates an inhibitory receptor on a B cell,e.g., an anti-FCRL antibody molecule, e.g., an agonistic anti-FCRLantibody molecule; a specific targeting moiety; or is absent; providedthat an effector binding moiety and a specific targeting moiety arepresent. In some embodiments, Linker A and Linker B comprise Fc moieties(e.g., self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In embodiment the anti-FCRL molecule comprises: an anti-FCRL antibodymolecule, e.g., an agonistic anti-FCRL antibody molecule, directed toFCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.

In some embodiments:

R1 and R3 each comprises an agonistic anti-FCRL antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In embodiment the anti-FCRL molecule comprises: an anti-FCRL antibodymolecule, e.g., an agonistic anti-FCRL antibody molecule directed toFCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.

In some embodiments:

R1 and R3 independently comprise specific targeting moieties, e.g.,antibody molecules against a tissue antigen; and

R2 and R4 each comprises an anti-FCRL antibody molecule, e.g., anagonistic anti-FCRL antibody molecule, e.g., an scFv molecule.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In embodiment the anti-FCRL molecule comprises: an anti-FCRL antibodymolecule, e.g., an agonistic anti-FCRL antibody molecule directed toFCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.

In some embodiments:

One of R1, R2, R3 and R4 comprises an anti-BCR antibody molecule, e.g.,an antagonistic anti-BCR antibody molecule, one comprises an anti FCRLantibody molecule, and one comprises a specific targeting moiety.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In some embodiments, the anti-FCRL molecule comprises: an anti-FCRLantibody molecule, e.g., an agonistic anti-FCRL antibody moleculedirected to FCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.

In some embodiments:

One of R1, R2, R3 and R4 comprises a bispecfic antibody moleculecomprising an anti-BCR antibody molecule, e.g., an antagonistic anti-BCRantibody molecule, and an anti FCRL antibody molecule, and one comprisesa specific targeting moiety;

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

In embodiment the anti-FCRL molecule comprises: an anti-FCRL antibodymolecule, e.g., an agonistic anti-FCRL antibody molecule directed toFCRL1, FCRL2, FCRL3, FCRL4, FCRL5, or FCRL6.

In some embodiments:

R1, R2, R3 and R4 each independently comprise:

i) an effector binding/modulating moiety, e.g., an ICIMbinding/modulating moiety, an IIC binding/modulating moiety, ICSMbinding/modulating moiety, or an SM binding/modulating moiety, thatminimizes or inhibits T cell activity, expansion, or function (a T celleffector binding/modulating moiety);

ii) an effector binding/modulating moiety, e.g., an ICIMbinding/modulating moiety, an IIC binding/modulating moiety, ICSMbinding/modulating moiety, or an SM binding/modulating moiety, thatminimizes or inhibits B cell activity, expansion, or function (a B celleffector binding/modulating moiety);

iii) a specific targeting moiety; or

iv) is absent;

provided that, a T cell effector binding/modulating moiety, a B celleffector binding/modulating moiety, and a specific targeting moiety arepresent.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties).

In some embodiments, one of R1, R2, R3, and R4 comprises an agonisticanti-PD-1 antibody and one comprises an HLA-G molecule.

In some embodiments, one of R1, R2, R3, and R4 comprises an SMbinding/modulating moiety, e.g., a CD39 molecule or a CD73 molecule. Insome embodiments, one of R1, R2, R3, and R4 comprises an entity thatbinds, activates, or maintains, a regulatory immune cell, e.g., a Tregcell or a Breg cell, for example, an IL-2 mutein molecule.

In some embodiments, one of R1, R2, R3, and R4 comprises an agonisticanti-PD-1 antibody, or one comprises an HLA-G molecule, and onecomprises an IL-2 mutein molecule. In some embodiments, the PD-1antibody is replaced with a IL-2 mutein molecule. In some embodiments,one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody, onecomprises an HLA-G molecule, and one comprises CD39 molecule or a CD73molecule. In some embodiments, the PD-1 antibody is replaced with a IL-2mutein molecule.

Linker Regions

As discussed elsewhere herein specific targeting and effectorbinding/modulating moieties can be linked by linker regions. Any linkerregion described herein can be used as a linker. For example, linkerRegions A and B can comprise Fc regions. In some embodiments, atherapeutic compound comprises a Linker Region that can self-associate.In some embodiments, a therapeutic compound comprises a Linker Regionthat has a moiety that minimizes self association, and typically LinkerRegion A and Linker Region B are heterodimers. Linkers also includeglycine/serine linkers. In some embodiments, the linker can comprise oneor more repeats of GGGGS (SEQ ID NO: 23). In some embodiments, thelinker comprises 1, 2, 3, 4, or 5 repeats of SEQ ID NO: 23. In someembodiments, the linker comprises or

(SEQ ID NO: 22) GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 30) GGGGSGGGGSGGGGS.These linkers can be used in any of the therapeutic compounds orcompositions provided herein.

The linker region can comprise a Fc region that has been modified (e.g.mutated) to produce a heterodimer. In some embodiments, the CH3 domainof the Fc region can be mutated. Examples of such Fc regions can befound in, for example, U.S. Pat. No. 9,574,010, which is herebyincorporated by reference in its entirety. The Fc region as definedherein comprises a CH3 domain or fragment thereof, and may additionallycomprise one or more addition constant region domains, or fragmentsthereof, including hinge, CH1, or CH2. It will be understood that thenumbering of the Fc amino acid residues is that of the EU index as inKabat et al., 1991, NIH Publication 91-3242, National TechnicalInformation Service, Springfield, Va. The “EU index as set forth inKabat” refers to the EU index numbering of the human IgG1 Kabatantibody. For convenience, Table B of U.S. Pat. No. 9,574,010 providesthe amino acids numbered according to the EU index as set forth in Kabatof the CH2 and CH3 domain from human IgG1, which is hereby incorporatedby reference. Table 1.1 of U.S. Pat. No. 9,574,010 provides mutations ofvariant Fc heterodimers that can be used as linker regions. Table 1.1 ofU.S. Pat. No. 9,574,010 is hereby incorporated by reference.

In some embodiments, the Linker Region A comprises a first CH3 domainpolypeptide and a the Linker Region B comprises a second CH3 domainpolypeptide, the first and second CH3 domain polypeptides independentlycomprising amino acid modifications as compared to a wild-type CH3domain polypeptide, wherein the first CH3 domain polypeptide comprisesamino acid modifications at positions T350, L351, F405, and Y407, andthe second CH3 domain polypeptide comprises amino acid modifications atpositions T350, T366, K392 and T394, wherein the amino acid modificationat position T350 is T350V, T3501, T350L or T350M; the amino acidmodification at position L351 is L351Y; the amino acid modification atposition F405 is F405A, F405V, F405T or F405S; the amino acidmodification at position Y407 is Y407V, Y407A or Y407I; the amino acidmodification at position T366 is T366L, T366I, T366V, or T366M, theamino acid modification at position K392 is K392F, K392L or K392M, andthe amino acid modification at position T394 is T394W, and wherein thenumbering of amino acid residues is according to the EU index as setforth in Kabat.

In some embodiments, the amino acid modification at position K392 isK392M or K392L. In some embodiments, the amino acid modification atposition T350 is T350V. In some embodiments, the first CH3 domainpolypeptide further comprises one or more amino acid modificationsselected from Q347R and one of S400R or S400E. In some embodiments, thesecond CH3 domain polypeptide further comprises one or more amino acidmodifications selected from L351Y, K360E, and one of N390R, N390D orN390E. In some embodiments, the first CH3 domain polypeptide furthercomprises one or more amino acid modifications selected from Q347R andone of S400R or S400E, and the second CH3 domain polypeptide furthercomprises one or more amino acid modifications selected from L351Y,K360E, and one of N390R, N390D or N390E. In some embodiments, the aminoacid modification at position T350 is T350V. In some embodiments, theamino acid modification at position F405 is F405A. In some embodiments,the amino acid modification at position Y407 is Y407V. In someembodiments, the amino acid modification at position T366 is T366L orT366I. In some embodiments, the amino acid modification at position F405is F405A, the amino acid modification at position Y407 is and Y407V, theamino acid modification at position T366 is T366L or T366I, and theamino acid modification at position K392 is K392M or K392L. In someembodiments, the first CH3 domain polypeptide comprises the amino acidmodifications T350V, L351Y, S400E, F405V and Y407V, and the second CH3domain polypeptide comprises the amino acid modifications T350V, T366L,N390R, K392M and T394W. In some embodiments, the first CH3 domainpolypeptide comprises the amino acid modifications T350V, L351Y, S400E,F405T and Y407V, and the second CH3 domain polypeptide comprises theamino acid modifications T350V, T366L, N390R, K392M and T394W. In someembodiments, the first CH3 domain polypeptide comprises the amino acidmodifications T350V, L351Y, S400E, F405S and Y407V, and the second CH3domain polypeptide comprises the amino acid modifications T350V, T366L,N390R, K392M and T394W. In some embodiments, the first CH3 domainpolypeptide comprises the amino acid modifications T350V, L351Y, S400E,F405A and Y407V, and the second CH3 domain polypeptide comprises theamino acid modifications T350V, L351Y, T366L, N390R, K392M and T394W. Insome embodiments, the first CH3 domain polypeptide comprises the aminoacid modifications Q347R, T350V, L351Y, S400E, F405A and Y407V, and thesecond CH3 domain polypeptide comprises the amino acid modificationsT350V, K360E, T366L, N390R, K392M and T394W. In some embodiments, thefirst CH3 domain polypeptide comprises the amino acid modificationsT350V, L351Y, S400R, F405A and Y407V, and the second CH3 domainpolypeptide comprises the amino acid modifications T350V, T366L, N390D,K392M and T394W. In some embodiments, the first CH3 domain polypeptidecomprises the amino acid modifications T350V, L351Y, S400R, F405A andY407V, and the second CH3 domain polypeptide comprises the amino acidmodifications T350V, T366L, N390E, K392M and T394W. In some embodiments,the first CH3 domain polypeptide comprises the amino acid modificationsT350V, L351Y, S400E, F405A and Y407V, and the second CH3 domainpolypeptide comprises the amino acid modifications T350V, T366L, N390R,K392L and T394W. In some embodiments, the first CH3 domain polypeptidecomprises the amino acid modifications T350V, L351Y, S400E, F405A andY407V, and the second CH3 domain polypeptide comprises the amino acidmodifications T350V, T366L, N390R, K392F and T394W.

In some embodiments, an isolated heteromultimer comprising aheterodimeric CH3 domain comprising a first CH3 domain polypeptide and asecond CH3 domain polypeptide, the first CH3 domain polypeptidecomprising amino acid modifications at positions F405 and Y407, and thesecond CH3 domain polypeptide comprising amino acid modifications atpositions T366 and T394, wherein: (i) the first CH3 domain polypeptidefurther comprises an amino acid modification at position L351, and (ii)the second CH3 domain polypeptide further comprises an amino acidmodification at position K392, wherein the amino acid modification atposition F405 is F405A, F405T, F405S or F405V; and the amino acidmodification at position Y407 is Y407V, Y407A, Y407L or Y407I; the aminoacid modification at position T394 is T394W; the amino acid modificationat position L351 is L351Y; the amino acid modification at position K392is K392L, K392M, K392V or K392F, and the amino acid modification atposition T366 is T366I, T366L, T366M or T366V, wherein the heterodimericCH3 domain has a melting temperature (Tm) of about 70.degree. C. orgreater and a purity greater than about 90%, and wherein the numberingof amino acid residues is according to the EU index as set forth inKabat.

In some embodiments, the Linker Region A comprises a first CH3 domainpolypeptide and a t Linker Region B comprises a second CH3 domainpolypeptide, wherein the first CH3 domain polypeptide comprising aminoacid modifications at positions F405 and Y407, and the second CH3 domainpolypeptide comprising amino acid modifications at positions T366 andT394, wherein: (i) the first CH3 domain polypeptide further comprises anamino acid modification at position L351, and (ii) the second CH3 domainpolypeptide further comprises an amino acid modification at positionK392, wherein the amino acid modification at position F405 is F405A,F405T, F405S or F405V; and the amino acid modification at position Y407is Y407V, Y407A, Y407L or Y407I; the amino acid modification at positionT394 is T394W; the amino acid modification at position L351 is L351Y;the amino acid modification at position K392 is K392L, K392M, K392V orK392F, and the amino acid modification at position T366 is T366I, T366L,T366M or T366V, wherein the heterodimeric CH3 domain has a meltingtemperature (Tm) of about 70 C. or greater and a purity greater thanabout 90%, and wherein the numbering of amino acid residues is accordingto the EU index as set forth in Kabat. In some embodiments, the aminoacid modification at position F405 is F405A. In some embodiments, theamino acid modification at position T366 is T366I or T366L. In someembodiments, the amino acid modification at position Y407 is Y407V. Insome embodiments, the amino acid modification at position F405 is F405A,the amino acid modification at position Y407 is Y407V, the amino acidmodification at position T366 is T366I or T366L, and the amino acidmodification at position K392 is K392L or K392M. In some embodiments,the amino acid modification at position F405 is F405A, the amino acidmodification at position Y407 is Y407V, the amino acid modification atposition T366 is T366L, and the amino acid modification at position K392is K392M. In some embodiments, the amino acid modification at positionF405 is F405A, the amino acid modification at position Y407 is Y407V,the amino acid modification at position T366 is T366L, and the aminoacid modification at position K392 is K392L. In some embodiments, theamino acid modification at position F405 is F405A, the amino acidmodification at position Y407 is Y407V, the amino acid modification atposition T366 is T366I, and the amino acid modification at position K392is K392M. In some embodiments, the amino acid modification at positionF405 is F405A, the amino acid modification at position Y407 is Y407V,the amino acid modification at position T366 is T366I, and the aminoacid modification at position K392 is K392L. In some embodiments, thefirst CH3 domain polypeptide further comprises an amino acidmodification at position 5400 selected from S400D and S400E, and thesecond CH3 domain polypeptide further comprises the amino acidmodification N390R. In some embodiments, the amino acid modification atposition F405 is F405A, the amino acid modification at position Y407 isY405V, the amino acid modification at position S400 is S400E, the aminoacid modification at position T366 is T366L, and the amino acidmodification at position K392 is K392M.

In some embodiments, the modified first and second CH3 domains arecomprised by an Fc construct based on a type G immunoglobulin (IgG). TheIgG can be an IgG1, IgG2, IgG3 or IgG4.

Other Linker Region A and Linger Region B comprising variant CH3 domainsare described in U.S. Pat. Nos. 9,499,634 and 9,562,109, each of whichis incorporated by reference in its entirety.

A Linker Region A and Linker Region B can be complementary fragments ofa protein, e.g., a naturally occurring protein such as human serumalbumin. In embodiments, one of Linker Region A and Linker Region Bcomprises a first, e.g., an N terminal fragment of the protein, e.g.,hSA, and the other comprises a second, e.g., a C terminal fragment ofthe protein, e.g., has. In an embodiment the fragments comprise an Nterminal and a C terminal fragment. In an embodiment the fragmentscomprise two internal fragments. Typically the fragments do not overlap.In an embodiment the First and second fragment, together, provide theentire sequence of the original protein, e.g., hSA. The first fragmentprovides a N terminus and a C terminus for linking, e.g., fusing, toother sequences, e.g., sequences of R1, R2, R3, or R4 (as definedherein).

The Linker Region A and the Linker Region B can be derived from albuminpolypeptide. In some embodiments, the albumin polypeptide is selectedfrom native human serum albumin polypeptide and human alloalbuminpolypeptide. The albumin polypeptide can be modified such that theLinker Region A and Linker Region B interact with one another to formheterodimers. Examples of modified albumin polypeptides are described inU.S. Pat. Nos. 9,388,231 and 9,499,605, each of which is herebyincorporated by reference in its entirety. Accordingly, provided hereinare multifunctional heteromultimer proteins of the formula R1-LinkerRegion A-R2 and R3-Linker Region B-R4, wherein the Linker Region A andLinker Region B form a heteromultimer. In some embodiments, the LinkerRegion A comprises a first polypeptide and the Linker Region B comprisesa second polypeptide; wherein each of said first and second polypeptidescomprises an amino acid sequence comprising a segment of an albuminpolypeptide selected from native human serum albumin polypeptide andhuman alloalbumin polypeptide; wherein said first and secondpolypeptides are obtained by segmentation of said albumin polypeptide ata segmentation site, such that the segmentation results in a deletion ofzero to 3 amino acid residues at the segmentation site; wherein saidfirst polypeptide comprises at least one mutation selected from A194C,L198C, W214C, A217C, L331C and A335C, and said second polypeptidecomprises at least one mutation selected from L331C, A335C, V343C,L346C, A350C, V455C, and N458C; and wherein said first and secondpolypeptides self-assemble to form a quasi-native structure of themonomeric form of the albumin polypeptide.

In some embodiments, the segmentation site resides on a loop of thealbumin polypeptide that has a high solvent accessible surface area(SASA) and limited contact with the rest of the albumin structure, b)the segmentation results in a complementary interface between thetransporter polypeptides. These segmentation sites are described, forexample, in U.S. Pat. No. 9,388,231, which is hereby incorporated byreference in its entirety.

In some embodiments, the first polypeptide comprises residues 1-337 orresidues 1-293 of the albumin polypeptide with one or more of themutations described herein. In some embodiments, the second polypeptidecomprises residues of 342-585 or 304-585 of the albumin polypeptide withone or more of the mutations described herein. In some embodiments, thefirst polypeptide comprises residues 1-339, 1-300, 1-364, 1-441, 1-83,1-171, 1-281, 1-293, 1-114, 1-337, or 1-336 of the albumin protein. Insome embodiments, the second polypeptide comprises residues 301-585,365-585, 442-585, 85-585, 172-585, 282-585, or 115-585, 304-585,340-585, or 342-585 of the albumin protein.

In some embodiments, the first and second polypeptide comprise theresidues of the albumin protein as shown in the table below. Thesequence of the albumin protein is described below.

First Second Polypeptide Polypeptide Residues Residues 1-300 301-5851-364 365-585 1-441 442-585 1-83   85-585 1-171 172-585 1-281 282-5851-114 115-585 1-339 340-585 1-337 342-585 1-293 304-585 1-336 342-585

In some embodiments, the first and second polypeptides comprise a linkerthat can form a covalent bond with one another, such as a disulfidebond. A non-limiting example of the linker is a peptide linker. In someembodiments, the peptide linker comprises GGGGS. The linker can be fusedto the C-terminus of the first polypeptide and the N-terminus of thesecond polypeptide. The linker can also be used to attach the moietiesdescribed herein without abrogating the ability of the linkers to form adisulfide bond. In some embodiments, the first and second polypeptidesdo not comprise a linker that can form a covalent bond. In someembodiments, the first and second polypeptides have the followingsubstitutions.

First Second Polypeptide Polypeptide Substitution Substitution A217CV343C L331C A350C A217C L346C W214C V343C A335C L346C L198C V455C A217CA335C A217C L331C L198C N458C A194C V455C

The sequence of the albumin polypeptide can be The sequence of humanalbumin is as shown, in the post-protein form with the N-terminalsignaling residues removed

(MKWVTFISLLFLFSSAYSRGVFRR) DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL (human albumin)

In some embodiments, the Linker Region A and the Linker Region B form aheterodimer as described herein.

In some embodiments, the polypetide comprises at the N-terminus anantibody comprised of F(ab′)2 on an IgG1 Fc backbone fused with scFvs onthe C-terminus of the IgG Fc backbone. In some embodiments, the IgG Fcbackbone is a IgG1 Fc backbone. In some embodiments, the IgG1 backboneis replaced with a IgG4 backbone, IgG2 backbone, or other similar IgGbackbone. The IgG backbones described in this paragraph can be usedthroughout this application where a Fc region is referred to as part ofthe therapeutic compound. Thus, in some embodiments, the antibodycomprised of F(ab′)2 on an IgG1 Fc backbone can be an anti-MAdCAMantibody or an anti-PD-1 antibody on an IgG1 Fc or any other targetingmoiety or effector binding/modulating moiety provided herein. In someembodiments, the scFV segments fused to the C-terminus could be ananti-PD-1 antibody, if the N-terminus region is an anti-MAdCAM antibody,or anti-MAdCAM antibody, if the N-terminus region is an anti-PD-1antibody. In this non-limiting example, the N-terminus can be thetargeting moiety, such as any one of the ones provided for herein, andthe C-terminus can be the effector binding/modulating moiety, such asany of the ones provided for herein. Alternatively, in some embodiments,the N-terminus can be the effector binding/modulating moiety, such asany one of the ones provided for herein, and the C-terminus can be thetargeting moiety, such as any of the ones provided for herein.

In some embodiments, the N-terminus can be the targeting moiety, such asany one of the ones provided for herein, and the C-terminus can be theeffector binding/modulating moiety, such as any of the ones provided forherein.

In some embodiments, the therapeutic compound comprises two polypeptidesthat homodimerize. In some embodiments, the N-terminus of thepolypeptide comprises an effector binding/modulating moiety that isfused to a human IgG1 Fc domain (e.g. CH2 and/or CH3 domains). In someembodiments, the C-terminus of the Fc domain is another linker that isfused to the targeting moiety. Thus, in some embodiments, the moleculecould be represented using the formula of R1-Linker A-Fc Region-LinkerB-R2, wherein R1 can be an effector binding/modulating moiety, R2 is atargeting moiety, Linker A and Linker B are independently linkers asprovided for herein. In some embodiments, Linker 1 and Linker 2 aredifferent.

In some embodiments, the molecule could be represented using the formulaof R1-Linker A-Fc Region-Linker B-R2, wherein R1 can be a targetingmoiety, R2 is an effector binding/modulating moiety, Linker A and LinkerB are independently linkers as provided for herein. In some embodiments,Linker A and Linker B are different. The linkers can be chosen from thenon-limiting examples provided for herein. In some embodiments, R1 andR2 are independently selected from F(ab′)2 and scFV antibody domains. Insome embodiments, R1 and R2 are different antibody domains. In someembodiments, the scFV is in the VL-VH domain orientation.

In some embodiments, the therapeutic compound is a bispecific antibody.In some embodiments, the bispecific antibodies are comprised of fourpolypeptide chains comprising the following:

Chain 1: nt-VH1-CH1-CH2-CH3-Linker A-scFv[VL2-Linker B-VH2]-ct

Chain 2: nt-VH1-CH1-CH2-CH3-Linker A-scFv[VL2-Linker B-VH2]-ct

Chain 3: nt-VL1-CL-ct

Chain 4: nt-VL1-CL-ct,

wherein chains 1 and 2 are identical to each other, and chains 3 and 4are identical to each other,

wherein chain 1 forms a homodimer with chain 2; and chain 3 and 4associate with chain 1 and chain 2. That is, when each light chainassociates with each heavy chain, VL1 associates with VH1 and CLassociates with CH1 to form two functional Fab units. Without beingbound to any particular theory, each scFv unit is intrinsicallyfunctional since VL2 and VH2 are covalently linked in tandem with alinker as provided herein (e.g. GGGGSG (SEQ ID NO: 23),GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22), or GGGGSGGGGSGGGGS (SEQ ID NO:30). The sequences of Linker A and Linker B, which are independent ofone another can be the same or different and as otherwise describedthroughout the present application. Thus, in some embodiments, Linker Acomprises GGGGS (SEQ ID NO: 23), or two repeats thereof, GGGGSGGGGSGGGGS(SEQ ID NO: 30), or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22). In someembodiments, Linker B comprises GGGGS (SEQ ID NO: 23), or two repeatsthereof, GGGGSGGGGSGGGGS (SEQ ID NO: 30), or GGGGSGGGGSGGGGSGGGGS (SEQID NO: 22). The scFv may be arranged in the NT-VH2-VL2-CT orNT-VL2-VH2-CT orientation. NT or nt stands for N-terminus and CT or ctstands for C-terminus of the protein. CH1, CH2, and CH3 are the domainsfrom the IgG Fc region, and CL stands for Constant Light chain, whichcan be either kappa or lambda family light chains. The other definitionsstand for the way they are normally used in the art.

In some embodiments, the VH1 and VL1 domains are derived from theeffector molecule and the VH2 and VL2 domains are derived from thetargeting moiety. In some embodiments the VH1 and VL1 domains arederived from a targeting moiety and the VH2 and VL2 domains are derivedfrom an effector binding/modulating moiety.

In some embodiments, the VH1 and VL1 domains are derived from ananti-PD-1 antibody, and the VH2 and VL2 domains are derived from ananti-MAdCAM antibody. In some embodiments the VH1 and VL1 domains arederived from an anti-MAdCAM antibody and the VH2 and VL2 domains arederived from an anti-PD-1 antibody.

In some embodiments, Linker A comprises 1, 2, 3, 4, or 5 GGGGS (SEQ IDNO: 23) repeats. In some embodiments, Linker B comprises 1, 2, 3, 4, or5 GGGGS (SEQ ID NO: 23) repeats. For the avoidance of doubt, thesequences of Linker A and Linker B, which are used throughout thisapplication, are independent of one another. Therefore, in someembodiments, Linker A and Linker B can be the same or different. In someembodiments, Linker A comprises GGGGS (SEQ ID NO: 23), or two repeatsthereof, GGGGSGGGGSGGGGS (SEQ ID NO: 30), or GGGGSGGGGSGGGGSGGGGS (SEQID NO: 22). In some embodiments, Linker B comprises GGGGS (SEQ ID NO:23), or two repeats thereof, GGGGSGGGGSGGGGS (SEQ ID NO: 30), orGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 22).

In some embodiments, the therapeutic compound comprises a light chainand a heavy chain. In some embodiments, the light and heavy chain beginat the N-terminus with the VH domain of a targeting moiety followed bythe CH1 domain of a human IgG1, which is fused to a Fc region (e.g.CH2-CH3) of human IgG1. In some embodiments, at the c-terminus of the Fcregion is fused to a linker as provided herein, such as but not limitedto, GGGGS (SEQ ID NO: 23), or two or three repeats thereof, orGGGGSGGGGSGGGGS (SEQ ID NO: 22). The linker can then be fused to aneffector binding/modulating moiety, such as any one of the effectormoieties provided for herein. The polypeptides can homodimerize becausethrough the heavy chain homodimerization, which results in a therapeuticcompound having two effector moieties, such as two anti-PD-1 antibodies.In this orientation, the targeting moiety is an IgG format, there aretwo Fab arms that each recognize binding partner of the targetingmoiety, for example, MAdCAM being bound by the anti-MAdCAM targetingmoiety.

In some embodiments, if the therapeutic compound comprises a Fc portion,the Fc domain, (portion) bears mutations to render the Fc region“effectorless,” that is unable to bind FcRs. The mutations that renderFc regions effectorless are known. In some embodiments, the mutations inthe Fc region, which is according to the known numbering system, areselected from the group consisting of: K322A, L234A, L235A, G237A,L234F, L235E, N297, P331S, or any combination thereof. In someembodiments, the Fc mutations comprises a mutation at L234 and/or L235and/or G237. In some embodiments, the Fc mutations comprise L234A and/orL235A mutations, which can be referred to as LALA mutations. In someembodiments, the Fc mutations comprise L234A, L235A, and G237Amutations.

Disclosed herein are Linker Region polypeptides, therapeutic peptides,and nucleic acids encoding the polypeptides (e.g. therapeuticcompounds), vectors comprising the nucleic acid sequences, and cellscomprising the nucleic acids or vectors

Therapeutic compounds can comprise a plurality of specific targetingmoieties. In some embodiments, the therapeutic compound comprises aplurality one specific targeting moiety, a plurality of copies of adonor specific targeting moiety or a plurality of tissue specifictargeting moieties. In some embodiments, a therapeutic compoundcomprises a first and a second donor specific targeting moiety, e.g., afirst donor specific targeting moiety specific for a first donor targetand a second donor specific targeting moiety specific for a second donortarget, e.g., wherein the first and second target are found on the samedonor tissue. In some embodiments, the therapeutic compound comprisese.g., a first specific targeting moiety for a tissue specific target anda second specific targeting moiety for a second target, e.g., whereinthe first and second target are found on the same or different targettissue,

In some embodiments, a therapeutic compound comprises a plurality ofeffector binding/modulating moieties each comprising an ICIMbinding/modulating moiety, the number of ICIM binding/modulatingmoieties is sufficiently low that clustering of the ICIMbinding/modulating moiety's ligand on immune cells (in the absence oftarget binding) is minimized, e.g., to avoid systemic agonizing ofimmune cells in the absence of binding of the therapeutic compound totarget.

Polypeptides Derived From Reference, e.g., Human Polypeptides

In some embodiments, a component of a therapeutic molecule is derivedfrom or based on a reference molecule, e.g., in the case of atherapeutic molecule for use in humans, from a naturally occurring humanpolypeptide. E.g., In some embodiments, all or a part of a CD39molecule, a CD73 molecule, a cell surface molecule binder, a donorspecific targeting moiety, an effector ligand binding molecule, an ICIMbinding/modulating moiety, an IIC binding/modulating moiety, aninhibitory immune checkpoint molecule ligand molecule, an inhibitorymolecule counter ligand molecule, a SM binding/modulating moiety, aspecific targeting moiety, a target ligand binding molecule, or a tissuespecific targeting moiety, can be based on or derived from a naturallyoccurring human polypeptide. E.g., a PD-L1 molecule can be based on orderived from a human PD-L1 sequence.

In some embodiments, a therapeutic compound component, e.g., a PD-L1molecule:

-   -   a) comprises all or a portion of, e.g., an active portion of, a        naturally occurring form of the human polypeptide;    -   b) comprises all or a portion of, e.g., an active portion of, a        human polypeptide having a sequence appearing in a database,        e.g., GenBank database, on Jan. 11, 2017, a naturally occurring        form of the human polypeptide that is not associated with a        disease state;    -   c) comprises a human polypeptide having a sequence that differs        by no more than 1, 2, 3, 4, 5, 10, 20, or 30 amino acid residues        from a sequence of a) or b);    -   d) comprises a human polypeptide having a sequence that differs        at no more than by 1, 2, 3, 4, 5 10, 20, or 30% its amino acids        residues from a sequence of a) or b);    -   e) comprises a human polypeptide having a sequence that does not        differ substantially from a sequence of a) or b); or    -   f) comprises a human polypeptide having a sequence of c), d),        or e) that does not differ substantially in a biological        activity, e.g., ability to enhance or inhibit an immune        response, from a human polypeptide having the sequence of a) or        b).

In some embodiments, therapeutic compounds can comprise a plurality ofeffector binding/modulating moieties. For example, a therapeuticcompound can comprise two or more of the following selected from:

(a) an ICIM binding/modulating moiety; (b) an IIC binding/modulatingmoiety; (c) an SM binding/modulating moiety, or (d) an ICSMbinding/modulating moiety. In some embodiments, for example, atherapeutic compound can comprise a plurality, e.g., two, ICIMbinding/modulating moieties (wherein they are the same or different); byway of example, two that activate or agonize PD-1; a plurality, e.g.,two, IIC binding/modulating moieties; (wherein they are the same ordifferent); a plurality, e.g., two, SM binding/modulating moieties(wherein they are the same or different), or a plurality, e.g., tow,ICSM binding/modulating moieties (wherein they are the same ordifferent). In some embodiments, the therapeutic compound can comprisean ICIM binding/modulating moiety and an IIC binding/modulating moiety;an ICIM binding/modulating moiety and an SM binding/modulating moiety;an IIC binding/modulating moiety and an SM binding/modulating moiety, anICIM binding/modulating moiety and an ICSM binding/modulating moiety; anIIC binding/modulating moiety and an ICSM binding/modulating moiety; oran ICSM binding/modulating moiety and an SM binding/modulating moiety.In some embodiments, the therapeutic compound comprises a plurality oftargeting moieties. In some embodiments, the targeting moieties can bethe same or different.

Pharmaceutical Compositions and Kits

In another aspect, the present embodiments provide compositions, e.g.,pharmaceutically acceptable compositions, which include a therapeuticcompound described herein, formulated together with a pharmaceuticallyacceptable carrier. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, isotonic andabsorption delaying agents, and the like that are physiologicallycompatible.

The carrier can be suitable for intravenous, intramuscular,subcutaneous, parenteral, rectal, local, ophthalmic, topical, spinal orepidermal administration (e.g. by injection or infusion). As usedherein, the term “carrier” means a diluent, adjuvant, or excipient withwhich a compound is administered. In some embodiments, pharmaceuticalcarriers can also be liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. The pharmaceuticalcarriers can also be saline, gum acacia, gelatin, starch paste, talc,keratin, colloidal silica, urea, and the like. In addition, auxiliary,stabilizing, thickening, lubricating and coloring agents can be used.The carriers can be used in pharmaceutical compositions comprising thetherapeutic compounds provided for herein.

The compositions and compounds of the embodiments provided for hereinmay be in a variety of forms. These include, for example, liquid,semi-solid and solid dosage forms, such as liquid solutions (e.g.,injectable and infusible solutions), dispersions or suspensions,liposomes and suppositories. The preferred form depends on the intendedmode of administration and therapeutic application. Typical compositionsare in the form of injectable or infusible solutions. In someembodiments, the mode of administration is parenteral (e.g.,intravenous, subcutaneous, intraperitoneal, intramuscular). In someembodiments, the therapeutic molecule is administered by intravenousinfusion or injection. In another embodiment, the therapeutic moleculeis administered by intramuscular or subcutaneous injection. In anotherembodiment, the therapeutic molecule is administered locally, e.g., byinjection, or topical application, to a target site. The phrases“parenteral administration” and “administered parenterally” as usedherein means modes of administration other than enteral and topicaladministration, usually by injection, and includes, without limitation,intravenous, intramuscular, intraarterial, intrathecal, intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid,intraspinal, epidural and intrasternal injection and infusion.

Therapeutic compositions typically should be sterile and stable underthe conditions of manufacture and storage. The composition can beformulated as a solution, microemulsion, dispersion, liposome, or otherordered structure suitable to high therapeutic molecule concentration.Sterile injectable solutions can be prepared by incorporating the activecompound (i.e., therapeutic molecule) in the required amount in anappropriate solvent with one or a combination of ingredients enumeratedabove, as required, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the active compound into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-dryingthat yields a powder of the active ingredient plus any additionaldesired ingredient from a previously sterile-filtered solution thereof.The proper fluidity of a solution can be maintained, for example, by theuse of a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prolonged absorption of injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

As will be appreciated by the skilled artisan, the route and/or mode ofadministration will vary depending upon the desired results. In certainembodiments, the active compound may be prepared with a carrier thatwill protect the compound against rapid release, such as a controlledrelease formulation, including implants, transdermal patches, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are patented orgenerally known to those skilled in the art. See, e.g., Sustained andControlled Release Drug Delivery Systems, J. R. Robinson, ed., MarcelDekker, Inc., New York, 1978.

In certain embodiments, a therapeutic compound can be orallyadministered, for example, with an inert diluent or an assimilableedible carrier. The compound (and other ingredients, if desired) mayalso be enclosed in a hard or soft shell gelatin capsule, compressedinto tablets, or incorporated directly into the subject's diet. For oraltherapeutic administration, the compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.To administer a compound by other than parenteral administration, it maybe necessary to coat the compound with, or co-administer the compoundwith, a material to prevent its inactivation. Therapeutic compositionscan also be administered with medical devices known in the art.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms are dictated by and directly dependent on (a)the unique characteristics of the active compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of a therapeutic compound is 0.1-30mg/kg, more preferably 1-25 mg/kg. Dosages and therapeutic regimens ofthe therapeutic compound can be determined by a skilled artisan. Incertain embodiments, the therapeutic compound is administered byinjection (e.g., subcutaneously or intravenously) at a dose of about 1to 40 mg/kg, e.g., 1 to 30 mg/kg, e.g., about 5 to 25 mg/kg, about 10 to20 mg/kg, about 1 to 5 mg/kg, 1 to 10 mg/kg, 5 to 15 mg/kg, 10 to 20mg/kg, 15 to 25 mg/kg, or about 3 mg/kg. The dosing schedule can varyfrom e.g., once a week to once every 2, 3, or 4 weeks. In oneembodiment, the therapeutic compound is administered at a dose fromabout 10 to 20 mg/kg every other week. The therapeutic compound can beadministered by intravenous infusion at a rate of more than 20 mg/min,e.g., 20-40 mg/min, and typically greater than or equal to 40 mg/min toreach a dose of about 35 to 440 mg/m2, typically about 70 to 310 mg/m2,and more typically, about 110 to 130 mg/m2. In embodiments, the infusionrate of about 110 to 130 mg/m2 achieves a level of about 3 mg/kg. Inother embodiments, the therapeutic compound can be administered byintravenous infusion at a rate of less than 10 mg/min, e.g., less thanor equal to 5 mg/min to reach a dose of about 1 to 100 mg/m2, e.g.,about 5 to 50 mg/m2, about 7 to 25 mg/m2, or, about 10 mg/m2. In someembodiments, the therapeutic compound is infused over a period of about30 min. It is to be noted that dosage values may vary with the type andseverity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimensshould be adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions, and that dosage ranges set forthherein are exemplary only and are not intended to limit the scope orpractice of the claimed composition.

The pharmaceutical compositions may include a “therapeutically effectiveamount” or a “prophylactically effective amount” of a therapeuticmolecule. A “therapeutically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired therapeutic result. A therapeutically effective amount of atherapeutic molecule may vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of thetherapeutic compound to elicit a desired response in the individual. Atherapeutically effective amount is also one in which any toxic ordetrimental effects of a therapeutic molecule t is outweighed by thetherapeutically beneficial effects. A “therapeutically effective dosage”preferably inhibits a measurable parameter, e.g., immune attack at leastabout 20%, more preferably by at least about 40%, even more preferablyby at least about 60%, and still more preferably by at least about 80%relative to untreated subjects. The ability of a compound to inhibit ameasurable parameter, e.g., immune attack, can be evaluated in an animalmodel system predictive of efficacy in transplant rejection orautoimmune disorders. Alternatively, this property of a composition canbe evaluated by examining the ability of the compound to inhibit, suchinhibition in vitro by assays known to the skilled practitioner.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Also within the scope of the embodiments is a kit comprising atherapeutic compound described herein. The kit can include one or moreother elements including: instructions for use; other reagents, e.g., alabel, a therapeutic agent, or an agent useful for chelating, orotherwise coupling, a therapeutic molecule to a label or othertherapeutic agent, or a radioprotective composition; devices or othermaterials for preparing the a therapeutic molecule for administration;pharmaceutically acceptable carriers; and devices or other materials foradministration to a subject.

In some embodiments, embodiments provided herein also include, but arenot limited to:

-   1. A therapeutic compound comprising:

i) a specific targeting moiety selected from:

-   -   a) a donor specific targeting moiety which, e.g., preferentially        binds a donor target; or    -   b) a tissue specific targeting moiety which, e.g.,        preferentially binds target tissue of a subject; and

ii) an effector binding/modulating moiety selected from:

-   -   (a) an immune cell inhibitory molecule binding/modulating moiety        (ICIM binding/modulating moiety);    -   (b) an immunosuppressive immune cell binding/modulating moiety        (IIC binding/modulating moiety); or    -   (c) an effector binding/modulating moiety that, as part of a        therapeutic compound, promotes an immuno-suppressive local        microenvironment, e.g., by providing in the proximity of the        target, a substance that inhibits or minimizes attack by the        immune system of the target (SM binding/modulating moiety).

-   2. The therapeutic compound of embodiment 1, wherein the effector    binding/modulating moiety directly binds and activates an inhibitory    receptor.

-   3. The therapeutic compound of embodiment 2, wherein the effector    binding/modulating moiety is an inhibitory immune checkpoint    molecule.

-   4. The therapeutic compound of any of embodiments 1-3, wherein the    effector binding/modulating moiety is expressed by an immune cell.

-   5. The therapeutic compound of embodiment 4, wherein the immune cell    contributes to an unwanted immune response.

-   6. The therapeutic compound of embodiments 4 or 5, wherein the    immune cell causes a disease pathology.

-   7. The therapeutic compound of embodiment 1, wherein the ability of    the therapeutic molecule to agonize the molecule to which the    effector binding/modulating binds is greater, e.g., 2, 5, 10, 100,    500, or 1,000 times greater, when the therapeutic compound is bound    to a target through the targeting moiety than when the therapeutic    compound is not bound to target through the targeting moiety.

-   8. The therapeutic compound of embodiments 1-7, wherein when binding    as a monomer (or binding when the therapeutic compound is not    multimerized), to its cognate ligand, e.g., an inhibitory immune    checkpoint molecule, does not agonize or substantially agonize, the    cognate ligand.

-   9. The therapeutic compound of embodiments 1-8, wherein at a    therapeutically effective dose of the therapeutic compound, there is    significant, systemic agonization of the molecule to which the    effector binding/modulating moiety binds.

-   10. The therapeutic compound of embodiments 1-9, wherein at a    therapeutically effective dose of the therapeutic compound, the    agonization of the molecule to which the effector binding/modulating    moiety binds occurs substantially only at a target site to which the    targeting moiety binds to.

-   11. The therapeutic compound of embodiments 1-9, wherein binding of    the therapeutic compound to its cognate ligand, e.g., an inhibitory    immune checkpoint molecule, does not inhibit, or does not    substantially inhibit, binding of an endogenous counter ligand to    the cognate ligand, e.g., an inhibitory immune checkpoint molecule.

-   12. The therapeutic compound of embodiments 1-11, wherein binding of    the effector binding/modulating moiety to its cognate ligand,    inhibits the binding of an endogenous counter ligand to the cognate    ligand of the effector binding/modulating moiety by less than 60,    50, 40, 30, 20, 10, or 5%.

-   14. The therapeutic compound of embodiments 1-11, wherein binding of    the effector binding/modulating moiety to the cognate ligand,    results in substantially no antagonism of the cognate ligand of the    effector binding/modulating molecule.

-   15. The therapeutic compound of embodiment 1, wherein the effector    binding/modulating moiety comprises an ICIM binding/modulating    moiety.

-   16. The therapeutic compound of embodiment 15, wherein the effector    binding/modulating moiety comprises an ICIM binding/modulating    moiety comprising an inhibitory immune checkpoint molecule ligand    molecule.

-   17. The therapeutic compound of embodiment 16, wherein the    inhibitory immune molecule counter-ligand molecule comprises a PD-L1    molecule.

-   18. The therapeutic compound of embodiment 15, wherein the ICIM is    wherein the inhibitory immune molecule counter ligand molecule    engages a cognate inhibitory immune checkpoint molecule selected    from PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4.

-   19. The therapeutic compound of embodiment 18, wherein the ICIM is    an antibody.

-   20. The therapeutic compound of embodiment 18, wherein the ICIM    comprises an antibody that binds to PD-1, KIR2DL4, LILRB1, LILRB, or    CTLA-4.

-   21. The therapeutic compound of embodiment 20, wherein the antibody    is an antibody that binds to PD-1.

-   22. The therapeutic compound of embodiment 20, wherein the antibody    is an antibody that binds to PD-1 and is a PD-1 agonist.

-   23. The therapeutic compound of embodiment 20, wherein the antibody    is an antibody that binds to PD-1 and is a PD-1 agonist when    tethered at a target site.

-   24. The therapeutic compound of embodiment 16, wherein the    inhibitory immune molecule counter-ligand molecule comprises a HLA-G    molecule.

-   25. The therapeutic compound of embodiment 15, wherein the ICIM is    wherein the inhibitory immune molecule counter ligand molecule    engages a cognate inhibitory immune checkpoint molecule selected    from PD-1, KIR2DL4, LILRB1, LILRB, or CTLA-4.

-   26. The therapeutic compound of embodiment 15, wherein the    inhibitory immune molecule counter ligand molecule engages a cognate    inhibitory immune checkpoint molecule selected from Table 1.

-   27. The therapeutic compound of embodiment 15, wherein when binding    as a monomer, to its cognate inhibitory immune checkpoint molecule,    does not agonize or substantially agonize the inhibitory immune    checkpoint molecule.

-   28. The therapeutic compound of embodiment 15, wherein the    inhibitory immune molecule counter ligand has at least 60, 70, 80,    90, 95, 99, or 100% homology with a naturally occurring inhibitory    immune checkpoint molecule ligand.

-   29. The therapeutic compound of embodiment 1, wherein the effector    binding/modulating moiety comprises a ICIM binding/modulating moiety    which comprises a functional antibody molecule to a cell surface    inhibitory molecule.

-   30. The therapeutic compound of embodiment 1, wherein the cell    surface inhibitory molecule is an inhibitory immune checkpoint    molecule.

-   31. The compound of embodiment 30, wherein the inhibitory immune    checkpoint molecule is selected from PD-1, KIR2DL4, LILRB1, LILRB2,    CTLA-4, or selected from Table 1.

-   32. The therapeutic compound of any of embodiments 1-31, wherein the    level of systemic immune suppression at a therapeutically effective    dose of the therapeutic compound, is less than that given by the    standard of care with a systemic immune suppressant (if relevant),    or is less than that given by an equimolar amount of free (not as a    component of a therapeutic compound), effector binding/modulating    molecule.

-   33. The therapeutic compound of embodiment 1-32, wherein the level    of systemic immune activation, e.g., at a therapeutically effective    dose of the therapeutic compound, is less than that given by a    equimolar amount of free (not as a component of a therapeutic    compound), effector binding/modulating molecule.

-   34. The therapeutic compound of any one of embodiments 1-33, further    comprising a second effector binding/modulating moiety.

-   35. The therapeutic compound of embodiment 34, wherein the second    effector binding/modulating moiety, binds a different target than    the effector binding/modulating moiety.

-   36. The therapeutic compound embodiments 34 or 35, wherein the    second effector binding/modulating moiety comprises a IIC    binding/modulating moiety.

The therapeutic compound embodiments 34 or 35, wherein the secondeffector binding/modulating moiety comprises an SM binding/modulatingmoiety.

-   37. The therapeutic compound of embodiment 1, wherein the effector    binding/modulating moiety comprises an IIC binding/modulating    moiety.-   38. The therapeutic compound of embodiment 1, wherein the effector    binding/modulating moiety comprises an IIC binding/modulating    moiety, which, increases, recruits or accumulates an    immunosuppressive immune cell at the target site.-   39. The therapeutic compound of embodiment 1, wherein the effector    binding/modulating moiety comprises a cell surface molecule binder    which binds or specifically binds, a cell surface molecule on an    immunosuppressive immune cell.-   40. The therapeutic compound of embodiment 1, wherein the effector    binding/modulating moiety comprises a cell surface molecule ligand    molecule that binds or specifically binds, a cell surface molecule    on an immunosuppressive immune cell.-   41. The therapeutic compound of embodiment 1, wherein the effector    binding/modulating moiety comprises an antibody molecule that binds    a cell surface molecule on an immunosuppressive immune cell.-   42. The therapeutic compound of any of embodiments 38-41, wherein    the immunosuppressive immune cell comprises a T regulatory cell,    such as a a Foxp3+CD25+ T regulatory cell.-   43. The therapeutic compound of any of embodiments 1-42, wherein the    effector binding/modulating moiety binds GARP, and e.g., comprises    an antibody molecule that binds GARP on GARP expressing    immunosuppressive cells, e.g., Tregs.-   44. The therapeutic compound of embodiment 1, wherein the effector    binding/modulating moiety comprises an SM binding/modulating moiety.-   45. The therapeutic compound of embodiment 44, wherein SM    binding/modulating moiety promotes an immuno-suppressive local    microenvironment.-   46. The therapeutic compound of any of embodiments 44 and 45,    wherein the effector molecule binding moiety increases the    availability, e.g., by increasing the local concentration or amount,    of a substance which inhibits immune cell function, e.g., a    substance that inhibits the activation of an immune cell or the    function of an activated immune cell.-   47. The therapeutic compound of any of embodiments 44-46, wherein    the effector molecule binding moiety binds and accumulate a soluble    substance, e.g., an endogenous or exogenous substance, having    immunosuppressive function.-   48. The therapeutic compound of any of embodiments 44-47, wherein    the effector molecule binding moiety decreases the availability,    e.g., by decreasing the local concentration or amount, or    sequestering, of a substance which promotes immune cell function,    e.g., a substance that promotes the activation of an immune cell or    the function of an activated immune cell.-   49. The therapeutic compound of any one of embodiments 44-48,    wherein SM binding/modulating moiety promotes an immuno-suppressive    local microenvironment, e.g., by providing in the proximity of the    target, a substance that inhibits or minimizes attack by the immune    system of the target.-   50. The therapeutic compound of any one of embodiments 44-49,    wherein the SM binding/modulating moiety comprises a molecule that    inhibits or minimizes attack by the immune system of the target.-   51. The therapeutic compound any one of embodiments 44-50, wherein    the SM binding/modulating moiety binds and/or accumulate a soluble    substance, e.g., an endogenous or exogenous substance having    immunosuppressive function.-   52. The therapeutic compound any one of embodiments 44-51, wherein    the SM binding/modulating moiety binds and/or inhibits, sequesters,    degrades or otherwise neutralizes a substance, e.g., a soluble    substance, typically and endogenous soluble substance, that promotes    immune attack.-   53. The therapeutic compound any one of embodiments 44-52, wherein    the effector molecule binding moiety decreases the availability of    ATP or AMP.-   54. The therapeutic compound any one of embodiments 44-53, wherein    SM binding/modulating moiety binds, or comprises, a substance, e.g.,    CD39 or CD73, that depletes a component that promotes immune    effector cell function, e.g., ATP or AMP.-   55. The therapeutic compound any one of embodiments 44-54, wherein    the SM binding/modulating moiety comprises a CD39 molecule.-   56. The therapeutic compound any one of embodiments 44-54, wherein    the SM binding/modulating moiety comprises a CD73 molecule.-   57. The therapeutic compound any one of embodiments 44-54, wherein    the SM binding/modulating moiety comprises an anti-CD39 molecule.-   58. The therapeutic compound any one of embodiments 44-54, wherein    the SM binding/modulating moiety comprises an anti-CD73 antibody    molecule.-   59. The therapeutic compound any one of embodiments 44-54, wherein    the effector molecule binding moiety comprises an immune-suppressive    substance, e.g. a fragment an immunosuppressive protein.-   60. The therapeutic compound any one of embodiments 44-54, wherein    SM binding/modulating moiety comprises alkaline phosphatase    molecule.-   61. The therapeutic compound of embodiment 1, wherein the compound    has the formula from N-terminus to C-terminus:

R1-Linker Region A-R2 or R3-Linker Region B-R4, wherein,

R1, R2, R3, and R4, each independently comprises an effectorbinding/modulating moiety, e.g., an ICIM binding/modulating moiety, anIIC binding/modulating moiety, or an SM binding/modulating moiety; aspecific targeting moiety; or is absent; provided that an effectorbinding/modulating moiety and a specific targeting moiety are present.

-   62. The therapeutic compound of embodiment 61, wherein each of    Linker Region A and Linker Region B comprises an Fc region.-   63. The therapeutic compound of embodiment 61, wherein one of R1 and    R2 is anti-PD-1 antibody and one of R1 and R2 is an anti-MAdCAM    antibody.-   64. The therapeutic compound of embodiment 61, wherein one of R1 is    anti-PD-1 antibody and one R2 is an anti-MAdCAM antibody.-   65. The therapeutic compound of embodiment 61, wherein one of R1 is    anti-MAdCAM antibody and one R2 is an anti-PD-1 antibody.-   66. The therapeutic compound of embodiment 61, wherein one of R3 and    R4 is anti-PD-1 antibody and one of R3 and R4 is an anti-MAdCAM    antibody.-   67. The therapeutic compound of embodiment 61, wherein one of R3 is    anti-PD-1 antibody and one R4 is an anti-MAdCAM antibody.-   68. The therapeutic compound of embodiment 61, wherein one of R3 is    anti-MAdCAM antibody and one R4 is an anti-PD-1 antibody.-   69. The therapeutic compound of any of embodiments 61-68, wherein    the linker is absent.-   70. The therapeutic compound of any of embodiments 61-68, wherein    the linker is a Fc region.-   71. The therapeutic compound of any of embodiments 61-68, wherein    the linker is a glycine/serine linker, such as 1, 2, 3, 4, or 5    repeats of GGGGS (SEQ ID NO: 23).-   72. The therapeutic compound of any of embodiments 61-68, wherein    the linker comprises a Fc region and a glycine/serine linker, such    as 1, 2, 3, 4, or 5 repeats of GGGGS (SEQ ID NO: 23).-   73. The therapeutic compound of any of embodiments 61-72, wherein    the PD-1 antibody is a PD-1 agonist.-   74. The therapeutic compound of embodiment 61, wherein:

R1 and R3 independently comprise a functional anti-PD-1 antibodymolecule (an agonist of PD-1); and R2 and R4 independently comprisespecific targeting moieties, e.g., scFv molecules against a tissueantigen.

-   75. The therapeutic compound of any of embodiments 73 and 74,    wherein:

R1 and R3 independently comprise specific targeting moieties, e.g., ananti-tissue antigen antibody; and R2 and R4 independently comprise afunctional anti-PD-1 antibody molecule (an agonist of PD-1).

-   76. The therapeutic compound of any of embodiments 73 and 74,    wherein:

R1, R2, R3 and R4 each independently comprise: an SM binding/modulatingmoiety which modulates, e.g., binds and inhibits, sequesters, degradesor otherwise neutralizes a substance, e.g., a soluble molecule thatmodulates an immune response, e.g., ATP or AMP, e.g., a CD39 molecule ora CD73 molecule; a specific targeting moiety; or is absent;

provided that an SM binding/modulating moiety and a specific targetingmoiety are present.

-   77. The therapeutic compound of embodiment 61, wherein:

R1 and R3 independently comprise a CD39 molecule or a CD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   78. The therapeutic compound of embodiment 77, wherein:

R1 and R3 each comprises a CD39 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   79. The therapeutic compound of embodiments 61 or 77, wherein:

R1 and R3 each comprises a CD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   80. The therapeutic compound of embodiment 61, wherein:

one of R1 and R3 comprises a CD39 molecule and the other comprises aCD73 molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   81. The therapeutic compound of embodiment 61, wherein:

R1, R2, R3 and R4 each independently comprise: an HLA-G molecule; aspecific targeting moiety; or is absent;

provided that an HLA-G molecule and a specific targeting moiety arepresent.

-   82. The therapeutic compound of embodiments 61 or 81, wherein:

R1 and R3 each comprise an HLG-A molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

-   83. The therapeutic compound of any of embodiments 81 and 82,    wherein:

R1 and R3 each comprise an agonistic anti-LILRB1 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

-   84. The therapeutic compound of any of embodiments 81 and 82,    wherein:

R1 and R3 each comprise an agonistic anti-KIR2DL4 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

In some embodiments, Linker A and Linker B comprise Fc moieties (e.g.,self pairing Fc moieties or Fc moieties that do not, or do notsubstantially self pair).

-   85. The therapeutic compound of any of embodiments 81-84, wherein:

R1 and R3 each comprise an agonistic anti-LILRB2 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   86. The therapeutic compound of any of embodiments 81-84, wherein:

R1 and R3 each comprise an agonistic anti-NKG2A antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   87. The therapeutic compound of any of embodiments 81-84, wherein:

one of R1 and R3 comprises a first moiety chosen from, and the othercomprises a different moiety chosen from: an antagonistic anti-LILRB1antibody molecule, an agonistic anti-KR2DL4 antibody molecule, and anagonistic anti-NKG2A antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   88. The therapeutic compound of any of embodiments 81-84, wherein:

one of R1 and R3 comprises an antagonistic anti-LILRB1 antibody moleculeand the other comprises an agonistic anti-KR2DL4 antibody molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   89. The therapeutic compound of any of embodiments 81-84, wherein:    one of R1 and R3 comprises an antagonistic anti-LILRB1 antibody    molecule and the other comprises an agonistic anti-NKG2A antibody    molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   89A. The therapeutic compound of any of embodiments 81-84 wherein:

R1, R2, R3 and R4 each independently comprise: an IL-2 mutein molecule;a specific targeting moiety; or is absent; and

provided that an IL-2 mutein molecule and a specific targeting moietyare present.

-   89B. The therapeutic compound of embodiment 89A, wherein:

R1 and R3 each comprise an IL-2 mutein molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   89C. The therapeutic compound of embodiments 89A or 89B, wherein:

one of R1 and R3 comprises a MAdCAM binding molecule, e.g., ananti-MAdCAM antibody molecule or a GITR binding molecule, e.g., ananti-GITR antibody molecule and the other comprises an IL-2 muteinmolecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   89D. The therapeutic compound of embodiments 89A or 89B, wherein:

one of R1 and R3 comprises a GARP binding molecule, e.g., an anti-GARPantibody molecule and the other comprises an IL-2 mutein molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   89E. The therapeutic compound of embodiments 89A or 89B, wherein:

one of R1 and R3 comprises a GARP binding molecule, e.g., an anti-GARPantibody molecule or a GITR binding molecule, e.g., an anti-GITRantibody molecule and the other comprises an IL-2 mutein molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   89F. The therapeutic compound of embodiments 89A or 89B, wherein:

one of R1 and R3 comprises a GARP binding molecule, e.g., an anti-GARPantibody molecule and the other comprises an IL-2 mutein molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   89G. The therapeutic compound of embodiments 89A or 89B, wherein:

one of R1 and R3 comprises a GITR binding molecule, e.g., an anti-GITRantibody molecule, and the other comprises an IL-2 mutein molecule; and

R2 and R4 independently comprise specific targeting moieties, e.g., scFvmolecules against a tissue antigen.

-   89H. The therapeutic compound of embodiment 1, wherein the compound    is a polypeptide or protein, wherein the polypeptide or protein    comprises a targeting moiety that binds to a target cell and an    effector binding/modulating moiety, wherein the effector    binding/modulating moiety is a IL-2 mutant polypeptide (IL-2    mutein).-   89I. The therapeutic compound of embodiment 89H, wherein the    targeting moiety comprises an antibody that binds to a target    protein on the surface of a target cell.-   89J. The therapeutic compound of embodiment 89I, wherein the    antibody is an antibody that binds to MAdCAM, OAT1 (SLC22A6), OCT2    (SLC22A2), FXYD2, TSPAN7, DPP6, HEPACAM2, TMEM27, or GPR119.-   89K. The therapeutic compound of embodiment 89I, wherein the IL-2    mutein binds to a receptor expressed by an immune cell.-   89L. The therapeutic compound of embodiment 89I, wherein the immune    cell contributes to an unwanted immune response.-   89M. The therapeutic compound of any of embodiments 89H-89L, wherein    the immune cell causes a disease pathology.-   89N. The therapeutic compound of any of embodiments 89H-89M, wherein    the targeting moiety comprises an anti-MAdCAM antibody.-   89O. The therapeutic compound of embodiment 89H, wherein the    compound has the formula from N-terminus to C-terminus:

R1-Linker Region A-R2 or R3-Linker Region B-R4, wherein,

-   -   R1, R2, R3, and R4, each independently comprises the effector        binding/modulating moiety, the targeting moiety, or is absent.

-   89P. The therapeutic compound of embodiment 89O, wherein each of    Linker Region A and Linker Region B comprises an Fc region.

-   89Q. The therapeutic compound of embodiments 89O or 89P or, wherein    one of R1 and R2 is the IL-mutein antibody and one of R1 and R2 is    an anti-MAdCAM antibody.

-   89R. The therapeutic compound of embodiments 89O, 89P, or 89Q,    wherein R1 is the IL-mutein and R2 is an anti-MAdCAM antibody.

-   89S. The therapeutic compound of embodiments 89O, 89P, or 89Q,    wherein one of R1 is anti-MAdCAM antibody and one R2 is an anti-PD-1    antibody.

-   89T. The therapeutic compound of embodiments 89O, 89P, or 89Q,    wherein one of R3 and R4 is the IL-2 mutein and one of R3 and R4 is    an anti-MAdCAM antibody.

-   89U. The therapeutic compound of embodiments 89O, 89P, or 89Q,    wherein R3 is the IL-2 mutein and R4 is an anti-MAdCAM antibody.

-   89V. The therapeutic compound of embodiments 89O, 89P, or 89Q,    wherein R3 is an anti-MAdCAM antibody and one R4 is the IL-2 mutein.

-   89W. The therapeutic compound of any of embodiments 89O-89W, wherein    the linker is absent.

-   89X. The therapeutic compound of any of embodiments 89O-89W, wherein    the linker is or comprises a Fc region.

-   89Y. The therapeutic compound of any of embodiments 89O-89W, wherein    the linker comprises a glycine/serine linker.

-   89X. The therapeutic compound of any of embodiments 89O-89W, wherein    the linker comprises a sequence of GGGGSGGGGSGGGGSGGGGS,    GGGGSGGGGSGGGGS, GGGGSGGGGS, or GGGGS.

-   89Y. The therapeutic compound of embodiment 89H, wherein the IL-2    mutein comprises a IL-2 sequence of SEQ ID NO: 6, wherein peptide    comprises a mutation at a position that corresponds to position 53,    56, 80, or 118 of SEQ ID NO: 6.

-   89Z. The therapeutic compound of any of embodiments 89H-89Z, wherein    the IL-2 mutein comprises a IL-2 sequence of SEQ ID NO: 6, wherein    peptide comprises a mutation at a position that corresponds to    position 53, 56, 80, or 118 of SEQ ID NO: 6.

-   89AA. The therapeutic compound of embodiment 89Y, wherein the    mutation is a L to I mutation at position 53, 56, 80, or 118.

-   89BB. The therapeutic compound of embodiment 89Z, wherein the    mutation is a L to I mutation at position 53, 56, 80, or 118.

-   89CC. The therapeutic compound of any of embodiments 89H-89BB,    wherein the IL-2 mutein further comprises a mutation at one or more    positions of 29, 31, 35, 37, 48, 69, 71, 74, 88, and 125    corresponding to those positions in SEQ ID NO: 6.

-   89DD. The therapeutic compound of any of embodiments 89H-89CC,    wherein the IL-2 mutein further comprises a mutation at one or more    of positions E15, H16, Q22, D84, E95, or Q126 or 1, 2, 3, 4, 5, or    each of positions E15, H16, Q22, D84, E95, or Q126 is wild-type.

-   89EE. The therapeutic compound of any of embodiments 89H-89DD,    wherein the mutation in the mutein is one or more of E15Q, H16N,    Q22E, D84N, E95Q, or Q126E.

-   89FF. The therapeutic compound of any of embodiments 89H-89EE,    wherein the mutein comprises a N29S mutation in SEQ ID NO: 6.

-   89GG. The therapeutic compound of any of embodiments 89H-89FF,    wherein the mutein comprises a Y31S or a Y51H mutation.

-   89HH. The therapeutic compound of any of embodiments 89H-89GG,    wherein the mutein comprises a K35R mutation.

-   89II. The therapeutic compound of any of embodiments 89H-89HH,    wherein the mutein comprises a T37A mutation.

-   89JJ. The therapeutic compound of any of embodiments 89H-89II,    wherein the mutein comprises a K48E mutation.

-   89KK. The therapeutic compound of any of embodiments 89H-89JJ,    wherein the mutein comprises a V69A mutation.

-   89LL. The therapeutic compound of any of embodiments 89H-89KK,    wherein the mutein comprises a N71R mutation.

-   89MM. The therapeutic compound of any of embodiments 89H-89LL,    wherein the mutein comprises a Q74P mutation.

-   89NN. The therapeutic compound of any of embodiments 89H-89MM,    wherein the mutein comprises a N88D or a N88R mutation.

-   89OO. The therapeutic compound of any of embodiments 89H-89NN,    wherein the mutein comprises a C125A or C125S mutation.

-   89PP. The therapeutic compound of any of embodiments 89H-89OO,    wherein the IL-2 mutein is fused or linked to a Fc peptide.

-   89PP1. The therapeutic compound of embodiment 89PP, wherein the Fc    peptide comprises a mutation at one or more of positions of L234,    L247, L235, L248, G237, and G250.

-   89PP2. The therapeutic compound of embodiment 89PP1, wherein the    mutation is L to A or G to A mutation.

-   89PP3. The therapeutic compound of embodiment 89PP1, wherein the Fc    peptide comprises L247A, L248A, and/or a G250A mutations (Kabat    numbering).

-   89PP4. The therapeutic compound of embodiment 89PP1, wherein the Fc    peptide comprises a L234A mutation, a L235A mutation, and/or a G237A    mutation (EU numbering).

-   89QQ. The therapeutic compound of embodiment 89H, wherein the    compound comprises a polypeptide comprising a first chain and a    second chain that form the polypeptide, wherein the first chain    comprises:

V_(H)-H_(c)-Linker-C₁, wherein V_(H) is a variable heavy domain thatbinds to the target cell with a V_(L) domain of the second chain; H_(c)is a heavy chain of antibody comprising CH1-CH2-CH3 domain, the Linkeris a glycine/serine linker, and C₁ is a IL-2 mutein fused or linked to aFc protein in either the N-terminal or C-terminal orientation; and

the second chain comprises:

V_(L)-L_(c), wherein V_(L) is a variable light chain domain that bindsto the target cell with the V_(H) domain of the first chain, and the Lcdomain is a light chain CK domain.

-   89QQ1. The therapeutic compound of embodiment 89QQ, wherein the VH    and VL domain are anti-MAdCAM variable domains that bind to MAdCAM    expressed on a cell.-   89QQ2. The therapeutic compound of embodiment 89QQ or 89QQ1, wherein    the IL-2 mutein comprises a mutation at a position that corresponds    to position 53, 56, 80, or 118 of SEQ ID NO: 6.-   89QQ3. The therapeutic compound of embodiment 89QQ2, wherein the    mutation is a L to I mutation at position 53, 56, 80, or 118.-   89QQ4. The therapeutic compound of embodiments 89QQ2 or 89QQ3,    wherein the mutein further comprises a mutation at a position that    corresponds to position 69, 75, 88, and/or 125, or any combination    thereof.-   89QQ5. The therapeutic compound of embodiments 89QQ2 or 89QQ3,    wherein the IL-2 mutein comprises a mutation selected from the group    consisting of: at one of L53I, L56I, L80I, and L118I and the    mutations of V69A, Q74P, N88D or N88R, and optionally C125A or    C125S.-   89QQ6. The therapeutic compound of embodiment 89QQ5, wherein the    IL-2 mutein comprises a L53I mutation.-   89QQ7. The therapeutic compound of embodiment 89QQ5, wherein the    IL-2 mutein comprises a L56I mutation.-   89QQ8. The therapeutic compound of embodiment 89QQ5, wherein the    IL-2 mutein comprises a L80I mutation.-   89QQ9. The therapeutic compound of embodiment 89QQ5, wherein the    IL-2 mutein comprises a L118I mutation.-   89QQ10. The therapeutic compound of embodiment 89QQ5, wherein the    IL-2 mutein does not comprises any other mutations.-   89QQ11. The therapeutic compound of any one of embodiments    89QQ-89QQ10, wherein the Fc protein comprises L247A, L248A, and    G250A mutations or a L234A mutation, a L235A mutation, and/or a    G237A mutation according to KABAT numbering.-   89QQ12. The therapeutic compound of any one of embodiments    89QQ-89QQ11, wherein the Linker comprises a sequence of    GGGGSGGGGSGGGGS or GGGGSGGGGSGGGGSGGGGS.-   89QQ13. The therapeutic compound of any one of embodiments    89QQ-89QQ11, wherein the polypeptide comprises a Fc peptide    comprising a sequence described herein.-   90. The therapeutic compound of any of embodiments 81-84, wherein:

one of R1, R2, R3 and R4 comprises an anti-BCR antibody molecule, e.g.,an antagonistic anti-BCR antibody molecule, one comprises an anti FCRLantibody molecule, and one comprises specific targeting moiety.

-   91. The therapeutic compound of embodiment 90, wherein:

the anti-FCRL molecule comprises: an anti-FCRL antibody molecule, e.g.,an agonistic anti-FCRL antibody molecule, directed to FCRL1, FCRL2,FCRL3, FCRL4, FCRL5, or FCRL6.

-   92. The therapeutic compound of any of embodiments 81-84, wherein:

R1, R2, R3 and R4 each independently comprise:

i) an effector binding/modulating moiety, e.g., an ICIMbinding/modulating moiety, an IIC binding/modulating moiety, or an SMbinding/modulating moiety, that minimizes or inhibits T cell activity,expansion, or function (a T cell effector binding/modulating moiety);

ii) an effector binding/modulating moiety, e.g., an ICIMbinding/modulating moiety, an IIC binding/modulating moiety, or an SMbinding/modulating moiety, that minimizes or inhibits B cell activity,expansion, or function (a B cell effector binding/modulating moiety);

iii) a specific targeting moiety; or

iv) is absent; provided that, a T cell effector binding/modulatingmoiety, a B cell effector binding/modulating moiety, and a specifictargeting moiety are present.

-   93. The therapeutic compound of embodiment 92, wherein:

one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody andone comprises an HLA-G molecule.

-   94. The therapeutic compound embodiments 92-93, wherein:

one of R1, R2, R3, and R4 comprises an SM binding/modulating moiety,e.g., a CD39 molecule or a CD73 molecule.

-   95. The therapeutic compound of any of embodiments 92-94, wherein:

one of R1, R2, R3, and R4 comprises an entity that binds, activates, ormaintains, a regulatory immune cell, e.g., a Treg cell or a Breg cell.

-   96. The therapeutic compound of any of embodiments 92-95, wherein:

one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody orone comprises an HLA-G molecule.

-   97. The therapeutic compound of embodiment 96, wherein:

one of R1, R2, R3, and R4 comprises an agonistic anti-PD-1 antibody, onecomprises an HLA-G molecule, and one comprises CD39 molecule or a CD73molecule.

-   98. The therapeutic compound of any of embodiments 1-97, wherein the    effector binding/modulating moiety comprises a polypeptide.-   99. The therapeutic compound of any of embodiments 1-98, wherein the    effector binding/modulating moiety comprises a polypeptide having at    least 5, 10, 20, 30, 40, 50, 150, 200 or 250 amino acid residues.-   100. The therapeutic compound of any of embodiments 1-99, wherein    the effector binding/modulating moiety has a molecular weight of 5,    10, 15, 20, or 40 Kd.-   101. The therapeutic compound of any of embodiments 1-100, wherein    the effector binding/modulating moiety does not comprise an    inhibitor of the expression of apolipoprotein CIII, protein kinase    A, Src kinase, or Beta 1 integrin.-   102. The therapeutic compound of any of embodiments 1-100, wherein    the effector binding/modulating moiety does not comprise an    inhibitor of the activity of apolipoprotein CIII, protein kinase A,    Src kinase, or Beta 1 integrin.-   103. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target a tissue    selected from lung, skin, pancreas, retina, prostate, ovary, lymph    node, adrenal gland, liver or gut tissue.-   104. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target tubular cells,    e.g., proximal tubular epithelial cells-   105. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target TIE-2, APN,    TEM4, TEM6, ICAM-1, nucleolin P2Z receptor, Trk-A, FLJ10849,    HSPA12B, APP, or OX-45.-   106. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target a luminally    expressed protein.-   107. The therapeutic compound of any of embodiments 1-101, wherein    the donor target does not comprise a heart specific target.-   108. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target lung tissue.-   109. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target kidney tissue.-   110. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically pancreas lung tissue.-   111. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target gut tissue.-   112. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target prostate    tissue.-   113. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target brain tissue.-   114. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target CD71.-   115. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target CD90.-   116. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target MAdCAM.-   117. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target albumin.-   118. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target carbonic    anhydrase IV.-   119. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target ZG16-p.-   120. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target dipeptidyl    peptidase IV.-   121. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target the luminal    surface of a vascular endothelial cell membrane.-   121. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target heart tissue.-   122. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target a tumor, solid    tumor, or the vascular of a solid tumor.-   123. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target skin tissue.-   124. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target epidermal    tissue.-   125. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target the basement    membrane.-   126. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target a Dsg    polypeptide.-   127. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target Dsg1.-   128. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target Dsg3.-   129. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target BP180.-   130. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not specifically target desmoglein.-   131. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not comprise a complement modulator,    e.g., a compliment inhibitor, such as, but not limited to, those    described in U.S. Pat. No. 8,454,963, which is hereby incorporated    by reference in its entirety.-   133. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not comprise an imaging agent.-   134. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not comprise an imaging agent selected    from the group of: a radioactive agent, a radioisotope, a    radiopharmaceutical, a contrast agent, a nanoparticle; an enzyme, a    prosthetic group, a fluorescent material, a luminescent material,    and a bioluminescent material, such as, but not limited to, those    described in U.S. Pat. No. 8,815,235, which is hereby incorporated    by reference in its entirety.-   135. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not comprise a radionuclide, such as,    but not limited to, those described in U.S. Pat. No. 6,232,287,    which is hereby incorporated by reference in its entirety.-   136. The therapeutic compound of any of embodiments 1-101, which is    not internalized by a donor cell to which it binds.-   137. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not enter the cell which is targeted    by the specific targeting moiety.-   138. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not kill the cell which is targeted by    the specific targeting moiety.-   139. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not enter the cell to which the    effector binding/modulating moiety binds.-   140. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not kill the cell to which the    effector binding/modulating moiety binds.-   141. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not comprise an autoantigenic peptide    or polypeptide.-   142. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not comprise an autoantigenic peptide    or polypeptide, e.g., does not comprise a peptide or polypeptide    against which the subject has autoantibodies.-   143. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not comprise an antibody molecule    derived from a mammal, e.g., a human, having an autoimmune disorder.-   144. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not comprise an antibody molecule    derived from a mammal, e.g., a human, having acute mucocutaneous PV.-   145. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not comprise an antibody molecule    derived from a mammal, e.g., a human, having Goodpasture's Disease-   146. The therapeutic compound of any of embodiments 1-101, wherein    the therapeutic compound does not comprise an antibody molecule    derived from a mammal, e.g., a human, having pemphigus vulgaris.-   141. The therapeutic compound of any of embodiments 1-146,    comprising a donor specific targeting moiety.-   142. The therapeutic compound of any of embodiments 141, that    localizes preferentially to an implanted donor tissue, as opposed to    tissue of a recipient.-   143. The therapeutic compound of embodiments 141-142, wherein, the    donor specific targeting moiety provides site-specific immune    privilege for a transplant tissue, e.g., an organ, from a donor.-   144. The therapeutic compound of embodiments 141-143, wherein the    donor specific targeting moiety binds to a product, e.g., a    polypeptide, of an allele present at a locus in the donor, which    allele is not present at the locus in the recipient-   145. The therapeutic compound of any of embodiments 141-144,    wherein, the donor specific targeting moiety preferentially binds to    an allele of a gene expressed on donor tissue, e.g., a transplant    tissue, e.g., an organ, as compared with an allele of the gene    expressed on subject tissue.-   146. The therapeutic compound of embodiments 141-145, wherein, the    donor specific targeting moiety has a binding affinity for an allele    of a gene expressed on donor tissue, e.g., a transplant tissue,    e.g., an organ, which is at least 2, 4, 5, 10, 50, 100, 500, 1,000,    5,000, or 10,000 fold greater than its affinity for an allele of the    gene expressed on subject tissue.-   147. The therapeutic compound of any of embodiments 141-146, wherein    the donor specific targeting moiety binds to the product, e.g., a    polypeptide, of an allele present at a locus in the donor, which    allele is not present at the locus in the recipient.-   148. The therapeutic compound of any one of embodiments 141-147,    wherein the binding is sufficiently specific that, e.g., at a    clinically effective dose of the therapeutic compound, unwanted,    substantial, or clinically unacceptable, systemic immune suppression    occurs.-   149. The therapeutic compound of any one of embodiments 141-148,    wherein the therapeutic compound accumulates at the target site,    e.g., binding of the donor specific targeting moiety to results in    accumulation of the therapeutic compound at the target site.-   150. The therapeutic compound of any one of embodiments 141-149,    wherein the donor specific targeting moiety binds a product of an    allele of a locus selected from Table 2, e.g., the HLA locus, e.g.,    the HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ or HLA-DR locus, which    allele is present in the donor but not the recipient. HLA-A, HLA-B,    HLA-C, HLA-DP, HLA-DQ or HLA-DR locus.-   151. The therapeutic compound of any one of embodiments 141-150,    wherein the donor specific targeting moiety binds an allele of HLA    A, an allele of HLA-B, an allele of HLA-C, an allele of HLA-DP, an    allele of HLA-, or an allele of HLA-.-   152. The therapeutic compound of any one of embodiments 141-151,    wherein the therapeutic compound is suitable for treating a subject    that has, will have, or is in need of, a transplant.-   153. The therapeutic compound of embodiment 152, wherein the    transplant comprises all or part of an organ, e.g., a liver, kidney,    heart, pancreas, thymus, skin or lung.-   154. The therapeutic compound of any one of embodiments 141-153,    wherein the donor specific targeting moiety comprises an antibody    molecule.-   155. The therapeutic compound of any one of embodiments 141-153,    wherein the donor specific targeting moiety comprises a target    specific binding polypeptide, or a target ligand binding molecule.-   156. The therapeutic compound of any one of embodiments 1-155,    comprising a tissue specific targeting moiety.-   157. The therapeutic compound of embodiment 156, wherein the tissue    specific targeting moiety is a molecule that specifically binds to    MAdCAM.-   158. The therapeutic compound of embodiment 156, wherein the tissue    specific targeting moiety is an antibody that specifically binds to    MAdCAM.-   159. The therapeutic compound of any one of embodiments, 156-158,    wherein the therapeutic compound is suitable for treating a subject    having, or is at risk, or elevated risk, for having, an autoimmune    disorder, e.g., an autoimmune disorder described herein.-   160. The therapeutic compound of any of embodiments 156-159, wherein    the therapeutic compound accumulates at the target site, e.g.,    binding of the tissue specific targeting moiety results in    accumulation of the therapeutic compound at the target site.-   161. The therapeutic compound of any of embodiments 156-160, wherein    the therapeutic compound which localizes, preferentially to a target    tissue, as opposed to other tissue of a subject.-   162. The therapeutic compound of any of embodiments 156-161, wherein    the therapeutic compound provides site-specific immune privilege for    a subject target tissue, e.g., a target tissue undergoing, or at    risk, or elevated risk, for, unwanted immune attack, e.g., in an    autoimmune disorder.-   163. The therapeutic compound of any of embodiments 156-161, wherein    the tissue specific targeting moiety, as a component of the    therapeutic compound, preferentially binds a subject target tissue    undergoing unwanted immune attack, e.g., in an autoimmune disorder.-   164. The therapeutic compound of any of embodiments 156-163, wherein    a tissue specific targeting moiety binds to the product, e.g., a    polypeptide, which is not present outside the target tissue, or is    present at sufficiently low levels that, at therapeutic    concentrations of therapeutic molecule, unacceptable levels of    immune suppression are absent or substantially absent.-   165. The therapeutic compound of any of embodiments 156-164,    wherein, the tissue specific targeting moiety binds a product, or    site on a product, which is more abundant in target tissue than in    non-target tissue.-   166. The therapeutic compound of any of embodiments 156-165,    wherein, therapeutic compound binds a product, or a site on a    product, that is present or expressed substantially exclusively on    target tissue.-   167 The therapeutic compound of any of embodiments 156-166, wherein    the product, or site on a product, to which the specific targeting    moiety binds, is sufficiently limited to the target tissue, that at    therapeutically effective level of therapeutic compound, the subject    does not suffer an unacceptable level, e.g., a clinically    significant level, of systemic immune suppression.-   168. The therapeutic compound of any of embodiments 156-167, wherein    the therapeutic compound, preferentially binds to a target tissue or    target tissue antigen, e.g., has a binding affinity for the target    tissue or antigen that is greater for target antigen or tissue,    e.g., at least 2, 4, 5, 10, 50, 100, 500, 1,000, 5,000, or 10,000    fold greater, than its affinity for than for non-target tissue or    antigen present outside the target tissue.-   169. The therapeutic compound of any of embodiments 156-168, wherein    the tissue specific targeting moiety binds to a product, e.g., a    polypeptide product, or site on a product, present at a preselected    site, e.g., a site of unwanted immune response in an autoimmune    disorder.-   170. The therapeutic compound of any of embodiments 156-169, wherein    therapeutic compound is suitable for the treatment of a subject    having, or at risk, or elevated risk, for having, type 1 diabetes.-   171. The therapeutic compound of any of embodiments 156-170, wherein    the target tissue comprises pancreatic tissue, e.g., pancreatic    islets or pancreatic beta cells, gut tissue (e.g. gut endothelial    cells), kidney tissue (e.g. kidney epithelial cells), or liver    tissue (e.g. liver epithelial cells).-   172. The therapeutic compound of any of embodiments 156-171, wherein    the effector binding/modulating moiety or targeting moiety binds a    polypeptide selected from those described herein, such as those    listed in Table 3, e.g., SEZ6L2, LRP11, DISP2, SLC30A8, FXYD2,    TSPAN7, or TMEM27.-   173. The therapeutic compound of any of embodiments 156-168, wherein    therapeutic compound is suitable for the treatment of a subject    having, or at risk, or elevated risk, for having, multiple    sclerosis.-   174. The therapeutic compound of embodiment 173, wherein the target    tissue comprises CNS tissue, myelin sheath, or myelin sheath of    oligodendrocytes.-   175. The therapeutic compound of any of embodiments 173-174, wherein    the effector binding/modulating moiety or targeting moiety binds a    polypeptide selected from those described herein and including, but    not limited to, Table 3, e.g., MOG, PLP, or MBP.-   176. The therapeutic compound of any of embodiments 156-168, wherein    therapeutic compound is suitable for the treatment of a subject    having, or at risk, or elevated risk, for having, cardiomyositis.-   177. The therapeutic compound of embodiment 176, wherein the target    tissue comprises cardiomyocytes, monocytes, macrophages, or myeloid    cells.-   178. The therapeutic compound of embodiments 176-177, wherein the    effector binding/modulating moiety binds or the targeting moiety a    polypeptide as described herein, including, but not limited to those    selected from Table 3, e.g., SIRPA (CD172a).-   179. The therapeutic compound of any of embodiments 156-168, wherein    therapeutic compound is suitable for the treatment of a subject    having, or at risk, or elevated risk, for having, inflammatory bowel    disease, autoimmune hepatitis (AIH); Primary Sclerosing Cholangitis    (PSC); Primary Biliary Sclerosis; (PBC); or transplant.-   180. The therapeutic compound of any of embodiments 156-168, wherein    the subject with has, is at risk or elevated risk for having Crohn's    disease or ulcerative colitis.-   181. The therapeutic compound of embodiments 179 or 180, wherein the    target tissue comprises gut cells, such as gut epithelial cells or    liver cells, such as liver epithelial cells.-   182. The therapeutic compound of embodiments 179-181, wherein the    effector binding/modulating moiety binds a polypeptide as described    herein, including, but not limited to those selected from Table 3,    e.g., PD-1.-   182. The therapeutic compound of embodiments 179-181, wherein the    targeting moiety binds a polypeptide as described herein, including,    but not limited to MAdCAM.-   183. The therapeutic compound of any of embodiments 156-168, wherein    therapeutic compound is suitable for the treatment of a subject    having, or at risk, or elevated risk, for having, rheumatoid    arthritis.-   184. The therapeutic compound of embodiment 183, wherein the target    tissue comprises cardiomyocytes, monocytes, macrophages, or myeloid    cells.-   185. The therapeutic compound of embodiments 183 or 184, wherein the    effector binding/modulating moiety or targeting moiety binds a    polypeptide selected from Table 3, e.g., SIRPA (CD172a).-   186. The therapeutic compound of any of embodiments 156-185, wherein    the tissue specific targeting moiety comprises an antibody molecule.-   187. The therapeutic compound of any of embodiments 156-185, wherein    the tissue specific targeting moiety comprises a target specific    binding polypeptide, or a target ligand binding molecule.-   188. The therapeutic compound of any of embodiments 156-185, wherein    the tissue specific targeting moiety comprises a target specific    binding polypeptide binds to MAdCAM.-   189. The therapeutic compound of any of embodiments 1-188, wherein    the therapeutic compound binds a cell surface molecule of an immune    effector cell, e.g., a T cell, B cell, NK cell, or other immune    cell, which cell propagates a pro-immune response.-   190. The therapeutic compound of any of embodiments 1-189, wherein    the therapeutic compound reduces the ability of an immune effector    cell, e.g., a T cell, B cell, NK cell, or other immune cell, to    propagate a pro-immune response.-   191. The therapeutic compound of any of embodiments 1-190, wherein    the specific targeting moiety targets a mammalian target, e.g., a    mammalian polypeptide, and the effector binding/modulating moiety    binds/modulates a mammalian immune component, e.g., a human immune    cell, e.g., a mammalian B cell, T cell, or macrophage.-   192. The therapeutic compound of any of embodiments 1-192, wherein    the specific targeting moiety targets a human target, e.g., a human    polypeptide, and the effector binding/modulating moiety    binds/modulates a human immune component, e.g., a human immune cell,    e.g., a human B cell, T cell, or macrophage.-   193. The therapeutic compound of any of embodiments 1-193, wherein    the therapeutic compound is configured for use in a human.-   194. The therapeutic compound of any of embodiments 1-191, wherein    the therapeutic compound is configured for use in a non-human    mammal.-   195. The therapeutic compound of any of embodiments 1-194, wherein    the therapeutic compound, e.g., the effector binding/modulating    moiety, comprises a PD-1 agonist.-   195.1. The therapeutic compound of any of the preceding embodiments,    wherein the therapeutic compound comprises a IL-2 mutein of SEQ ID    NO: 15, wherein the mutein comprises a mutation at position 73, 76,    100, or 138.-   195.2. The therapeutic compound of embodiment 195.1, wherein the    mutation is a L to I mutation at position 73, 76, 100, or 138.-   195.3. The therapeutic compound of embodiments 195.1 or 195.2,    wherein the IL-2 mutein further comprises a mutation at one or more    of positions 49, 51, 55, 57, 68, 89, 91, 94, 108, and 145.-   195.4. The therapeutic compound of any of embodiments 195.1-195.3,    wherein the mutein further comprises a mutation at one or more of    positions E35, H36, Q42, D104, E115, or Q146 or 1, 2, 3, 4, 5, or    each of E35, H36, Q42, D104, E115, or Q146 is wild-type.-   195.5. The therapeutic compound of embodiment 195.4, wherein the    mutation is one or more of E35Q, H36N, Q42E, D104N, E115Q, or Q146E.-   195.6. The therapeutic compound of any one of embodiments    195.1-195.5, wherein the IL-2 mutein comprises a N49S mutation.-   195.7. The therapeutic compound of any one of embodiments    195.1-195.6, wherein the IL-2 mutein comprises a Y51S or a Y51H    mutation.-   195.8. The therapeutic compound of any one of embodiments    195.1-195.7, wherein the IL-2 mutein comprises a K55R mutation.-   195.9. The therapeutic compound of any one of embodiments    195.1-195.8, wherein the IL-2 mutein comprises a T57A mutation.-   195.10. The therapeutic compound of any one of embodiments    195.1-195.8, wherein the IL-2 mutein comprises a K68E mutation, V89A    (V69A) mutation, a N91R (N71R) mutation, a Q94P or Q74P mutation, a    (N88D) or a N108R (N88R) mutation, a C145A(C125A) or C145S (C125 S)    mutation.-   195.11. The therapeutic compound of any one of embodiments    195.1-195.10, wherein the therapeutic compound comprises a IL-2    mutein of SEQ ID NO: 6, wherein the mutein comprises a mutation at    position 53, 56, 80, or 118 and one or more of the mutations recited    in embodiments 195.1-195.10.-   195.12 The therapeutic compound of any one of embodiments    195.1-195.11, wherein the IL-2 mutein is fused or linked to a Fc    peptide.-   195.13 The therapeutic compound of embodiment 195.12 wherein the Fc    peptide comprise a mutation at one or more of positions of L234,    L247, L235, L248, G237, and G250 (EU numbering).-   196. A method of treating a subject with inflammatory bowel disease,    the method comprising administering a therapeutic compound of any of    embodiments 1-195.13.13 to the subject to treat the inflammatory    bowel disease.-   197. The method of embodiment 196, wherein the subject with    inflammatory bowel disease has Crohn's disease.-   198. The method of embodiment 196, wherein the subject with    inflammatory bowel disease has ulcerative colitis.-   199. A method of treating a subject with auto-immune hepatitis, the    method comprising administering a therapeutic compound of any of    embodiments 1-195.13 to the subject to treat the auto-immune    hepatitis.-   200. A method of treating primary sclerosing cholangitis the method    comprising administering a therapeutic compound of any of    embodiments 1-195.13 to the subject to treat the primary sclerosing    cholangitis.-   201. A method of treating Type 1 diabetes the method comprising    administering a therapeutic compound of any of embodiments 1-195.13,    thereby treating the subject to treat the Type 1 diabetes.-   202. A method of treating a transplant subject comprising    administering a therapeutically effective amount of a therapeutic    compound of any of embodiments 1-195.13 to the subject, thereby    treating a transplant (recipient) subject.-   203. A method of treating GVHD in a subject having a transplanted a    donor tissue comprising administering a therapeutically effective    amount of a therapeutic compound of any of embodiments 1-195.13 to    the subject.-   204. The method of embodiment 203, wherein the therapeutic compound    is administered to the subject: prior to receiving the transplant;    prior to developing a symptom of GVHD; after or concurrent with    receiving the transplant; or after or concurrent with developing a    symptom of GVHD.-   205. A method of treating a subject having, or at risk, or elevated    risk, for having, an autoimmune disorder, comprising administering a    therapeutically effective amount of a therapeutic compound of any    embodiments 1-195.13, thereby treating the subject.-   206. The method of embodiment 205, wherein the subject has received,    will receive, or is in need of, allograft donor tissue.-   207. The method of any of embodiments 205-206, wherein the donor    tissue comprises a solid organ, e.g., a liver, kidney, heart,    pancreas, thymus, or lung.-   208. The method of any of embodiments 205-206, wherein the donor    tissue comprises all or part of an organ, e.g., a liver, kidney,    heart, pancreas, thymus, or lung.-   209. The method of any of embodiments 205-206, wherein the donor    tissue comprises skin.-   210. The method of any of embodiments 205-206, wherein the donor    tissue does not comprises skin.-   211. The method of any of embodiments 205-210, wherein the donor    tissue presents or expresses a product of an allele of a locus    locus, which allele is not present or expressed in the subject.-   212. The method of any of embodiments 205-210, wherein the donor    tissue presents or expresses a product of an allele of a locus    selected from Table 2, e.g., the HLA locus, e.g., the HLA-A, HLA-B,    HLA-C, HLA-DP, HLA-DQ or HLA-DR locus, which allele is not present    or expressed in the subject.-   213 The method of any of embodiments 205-212, comprising introducing    the transplant tissue into the subject.-   214. The method of any of embodiments 196-213, comprising monitoring    the subject for immune cell inactivation (e.g., to monitor unwanted    agonization of an immune inhibitory checkpoint molecule) at a site    distant from the target site, e.g., in the peripheral circulation or    the lymphatic system.-   215. The method of any of embodiments 196-214, comprising monitoring    the subject for immune cell activation (e.g., to monitor unwanted    antagonization of an immune inhibitory checkpoint molecule) at a    site distant from the target site, e.g., in the peripheral    circulation or the lymphatic system.-   216. The method of any of embodiments 196-215, wherein responsive to    the result of monitoring, selecting a course of treatment for the    subject, e.g., increasing the dose of the therapeutic compound,    decreasing the dose of the therapeutic compound, continuing    treatment with the therapeutic compound without a change in dose.-   217. The method of any of embodiments 196-216, comprising    administering the compound of embodiments 1-195.13, to the    recipient.-   218. The method of any of embodiments 196-216, wherein administering    comprises systemic administration, e.g., to the peripheral    circulatory system.-   219. The method of any of embodiments 196-216, wherein administering    comprises local administration, e.g., to the target tissue, the    donor tissue or the site of at which the target tissue or the donor    tissue is, or will be located.-   220. The method of any of embodiments 219, comprising administering    the therapeutic compound to the recipient prior to introduction of    the donor tissue into the recipient.-   221. The method of any of embodiments 219, comprising administering    the therapeutic compound, to the recipient after introduction of the    donor tissue into the recipient.-   222. The method of any of embodiments 213, comprising administering    the therapeutic compound to the recipient concurrent with    introduction of the donor tissue into the recipient.-   223. The method of embodiment 213, comprising contacting the    therapeutic compound with the donor tissue prior to introduction of    the donor tissue into the recipient.-   224. The method of any of embodiments 213, comprising providing the    therapeutic compound to the subject, wherein the transplant tissue    has been contacted with therapeutic compound prior to introduction    into the subject.-   225. The method of any of embodiments 213, comprising contacting the    therapeutic compound with the donor tissue after introduction of the    donor tissue into the recipient, e.g., by local administration to    the donor tissue.-   226. The method of any of embodiments 196-226, comprising    administering a therapeutic compound as provided for herein such    that therapeutic levels are present for at least 1, 5, 10, 14, or 28    days, for example, consecutive or non-consequitive days.-   227. The method of any of embodiments 196-226, wherein the subject    does not receive a non-targeted immune suppressive agent.-   228. The method of any of embodiments 196-226, wherein for the    subject has not received a non-targeted immune suppressive agent for    at least 1, 15, 30, 60, or 90 days prior to the initial    administration of the therapeutic compound.-   229. The method of any of embodiments 213, wherein the subject has    not received a non-targeted immune suppressive agent for at least 1,    15, 30, 60, or 90 days prior to introduction of the transplant    tissue.-   230. The method of any of embodiments 196-229, wherein the subject    does not receive a non-targeted immune suppressive agent for at    least 1, 15, 30, 60, 90, or 180 days after the initial    administration of the therapeutic compound.-   231. The method of any of embodiments 196-229, wherein the subject    does not receive a non-targeted immune suppressive agent for at    least 1, 15, 30, 60, 90, or 180 days after introduction of the    transplant tissue.-   232. The method of any of embodiments 196-231, comprising    administering a non-targeted immune suppressive agent to the    subject.-   233. The method of any of embodiments 196-232, wherein for the    subject receives a non-targeted immune suppressive agent for at    least 1, 15, 30, 60, or 90 days prior to the initial administration    of the therapeutic compound.-   234. The method of embodiment 213, wherein the subject receives a    non-targeted immune suppressive agent for at least 1, 15, 30, 60, or    90 days prior to introduction of the transplant tissue.-   235. The method of embodiment 234, wherein the subject receives a    non-targeted immune suppressive agent for at least 1, 15, 30, 60, 90    or 180 days after the initial administration of the therapeutic    compound.-   236. The method of any of embodiments 196-235, wherein the subject    receives a non-targeted immune suppressive agent for at least 1, 15,    30, 60, 90 or 180 days after introduction of the transplant tissue.-   237. The method of any of embodiments 196-235, wherein for the    subject receives a non-targeted immune suppressive agent prior to    the initial administration of the therapeutic compound but for no    more than 1, 15, 30, 60, 90 or 180 days.-   238. The method of embodiment 213, wherein the subject receives a    non-targeted immune suppressive agent prior to introduction of the    transplant tissue but for no more than 1, 15, 30, 60, 90 or 180    days.-   239. The method of any of embodiments 196-238, wherein the subject    receives a non-targeted immune suppressive agent after the initial    administration of the therapeutic compound but for no more than 1,    15, 30, 60, 90 or 180 days.-   240. The method of embodiment 213, wherein the subject receives a    non-targeted immune suppressive agent after introduction of the    transplant tissue but for no more than 1, 15, 30, 60, 90 or 180    days.-   241. The method of embodiment 213, wherein the subject is monitored    for rejection of the transplant tissue.-   242. The method of any of embodiments 196-242, a dosage of a    non-targeted immune suppressive agent is selected, or wherein    responsive to the monitoring, a dosage of a non-targeted immune    suppressive agent is selected.-   243. The method of embodiment 242, wherein the dosage is    administered.-   244. The method of embodiment 243, wherein the selected dosage is    zero, i.e., a non-targeted immune suppressive agent is not    administered.-   245. The method of embodiment 243, wherein the selected dosage is    non-zero, i.e., a non-targeted immune suppressive agent is    administered.-   246. The method of embodiment 243, wherein the dosage is less than    what would be administered in the absence of administration of a    therapeutic compound.-   247. The method of any of embodiments 196-246, wherein the subject    is a mammal, e.g., a non-human mammal.-   248. The method of any of embodiments 196-246, wherein the subject    is a human.-   249. The method of embodiment 213, wherein the donor and subject are    mismatched at an HLA locus, e.g., a major or minor locus.-   250. The method of embodiment 249, wherein the subject is a mammal,    e.g., a non-human mammal.-   251. The method of embodiment 249, wherein the subject is a human.-   252. A method of treating a subject having, or at risk, or elevated    risk, for having, an autoimmune disorder, comprising administering a    therapeutically effective amount of a therapeutic compound of any    embodiments 1-195.13, thereby treating the subject.-   253. The method of embodiment 252, wherein provision of the    therapeutic compound is initiated prior to the onset, or prior to    identification of onset, of symptoms of the autoimmune disorder.-   254. The method of any of embodiments 252-253, wherein provision of    the therapeutic compound is initiated after onset, or after    identification of onset, of symptoms of the autoimmune disorder.-   255. The method of embodiments 252-254, wherein autoimmune disorder    comprises type 1 diabetes.-   256. The therapeutic compound of any of embodiments 252-255, wherein    the target tissue comprises pancreatic islets or pancreatic beta    cells, gut tissue (e.g. gut endothelial cells), kidney tissue (e.g.    kidney epithelial cells), or liver tissue (e.g. liver epithelial    cells).-   257. The therapeutic compound of any of embodiments 252-256, wherein    the effector binding/modulating moiety or targeting moiety binds a    polypeptide selected from Table 3, e.g., MAdCAM, OAT1, OCT, DPP6,    SEZ6L2, LRP11, DISP2, SLC30A8, FXYD2, TSPAN7, or TMEM27 polypeptide.-   258. The method of any of embodiments 252-257, wherein provision of    the therapeutic compound is initiated prior to the onset, or prior    to identification of onset, of symptoms of type 1 diabetes.-   259. The method of any of embodiments 252-258, wherein provision of    the therapeutic compound is initiated prior to, or prior to    identification of the subject having a preselected characteristic or    symptom.-   260. The method of any of embodiments 252-259, wherein provision of    the therapeutic compound is initiated after onset, or after    identification of onset, of symptoms of type 1 diabetes.-   261. The method of any of embodiments 252-260, wherein provision of    the therapeutic compound is initiated after, or after identification    of the subject having a preselected characteristic or symptom.-   262, The method of any of embodiments 252-261, wherein the    therapeutic compound is a therapeutic compound of any of embodiments    1-195.13-   263 The method of any of embodiments 252-257, wherein therapeutic    compound is suitable for the treatment of a subject having, or at    risk, or elevated risk, for having, multiple sclerosis.-   264. The method of embodiment 263, wherein the target tissue    comprises CNS tissue, myelin sheath, or myelin sheath of    oligodendrocytes.-   265. The method of any of embodiments 263 or 264, wherein the    effector binding/modulating moiety or targeting moiety binds a    polypeptide selected from Table 3, e.g., a MOG, PLP, or MBP    polypeptide.-   266. The method of any of embodiments 263-265, wherein provision of    the therapeutic compound is initiated prior to the onset, or prior    to identification of onset, of symptoms of multiple sclerosis.-   267. The method of any of embodiments 263-265, wherein provision of    the therapeutic compound is initiated prior to, or prior to    identification of the subject a preselected characteristic or    symptom.-   268. The method of any of embodiments 263-265, wherein provision of    the therapeutic compound is initiated after onset, or after    identification of onset, of symptoms of multiple sclerosis.-   269. The method of any of embodiments 263-265, wherein provision of    the therapeutic compound is initiated after, or after identification    of the subject having a preselected characteristic or symptom.-   270. The method of any of embodiments 263-269, wherein the    therapeutic compound is a therapeutic compound of any of embodiments    1-195.13-   271. The method of any of embodiments 252-257, wherein the    therapeutic compound is suitable for the treatment of a subject    having, or at risk, or elevated risk, for having, cardiomyositis.-   272. The method of embodiment 271, wherein the target tissue    comprises cardiomyocytes, monocytes, macrophages, or myeloid cells.-   273. The method of embodiments 271 or 272, wherein the effector    binding/modulating moiety or targeting moiety binds a polypeptide    selected from Table 3, e.g., a SIRPA (CD172a) polypeptide.-   274. The method of any of embodiments 271-273, wherein provision of    the therapeutic compound is initiated prior to the onset, or prior    to identification of onset, of symptoms of cardiomyositis.-   275. The method of any of embodiments 271-273, wherein provision of    the therapeutic compound is initiated prior to, or prior to    identification of the subject having a preselected characteristic or    symptom.-   276. The method of any of embodiments 271-273, wherein provision of    the therapeutic compound is initiated after onset, or after    identification of onset, of symptoms of cardiomyositis.-   277. The method of any of embodiments 271-273, wherein provision of    the therapeutic compound is initiated after, or after identification    of the subject having a preselected characteristic or symptom.-   278. The method of any of embodiments 271-277, wherein the    therapeutic compound is a therapeutic compound of any of embodiments    1-195.13.-   279. The method of any of embodiments 252-257, wherein therapeutic    compound is suitable for the treatment of a subject having, or at    risk, or elevated risk, for having, rheumatoid arthritis.-   280. The method of embodiment 279, wherein the target tissue    comprises cardiomyocytes, monocytes, macrophages, or myeloid cells.-   281. The method of embodiments 279 or 280, wherein the effector    binding/modulating moiety or targeting moiety binds a polypeptide    selected from Table 3, e.g., a SIRPA (CD172a) polypeptide.-   282. The method of embodiments 279-281, wherein provision of the    therapeutic compound is initiated prior to the onset, or prior to    identification of onset, of symptoms of rheumatoid arthritis.-   283. The method of embodiments 279-281, wherein provision of the    therapeutic compound is initiated prior to, or prior to    identification of the subject having a preselected characteristic or    symptom.-   284. The method of embodiments 279-281, wherein provision of the    therapeutic compound is initiated after onset, or after    identification of onset, of symptoms of rheumatoid arthritis.-   285. The method of embodiments 279-281, wherein provision of the    therapeutic compound is initiated after, or after identification of    the subject having a preselected characteristic or symptom.-   286. The method of embodiments 279-285, wherein the therapeutic    compound is a therapeutic compound of any of embodiments 1-195.13.-   287. The method of any of embodiments 196-286, comprising monitoring    the subject for immune cell inactivation (e.g., to monitor unwanted    agonization of an immune inhibitory checkpoint molecule) at a site    distant from the target site, e.g., in the peripheral circulation or    the lymphatic system.-   288. The method of any of embodiments 196-287, comprising monitoring    the subject for immune cell activation (e.g., to monitor unwanted    antagonization of an immune inhibitory checkpoint molecule) at a    site distant from the target site, e.g., in the peripheral    circulation or the lymphatic system.-   289. The method of any of embodiments 196-288, wherein responsive to    the result of monitoring, selecting a course of treatment for the    subject, e.g., increasing the dose of the therapeutic compound,    decreasing the dose of the therapeutic compound, continuing    treatment with the therapeutic compound without a change in dose.-   290. The method of any of embodiments 196-289, wherein the subject    monitored for autoimmune attack of the target tissue.-   291. The method of embodiment 290, wherein responsive to the    monitoring, a dosage of the therapeutic compound is selected.-   292. The method of embodiment 291, wherein the dosage is    administered.-   293. The method of embodiment 290, wherein the selected dosage is    zero, i.e., administration of therapeutic compound is ceased.-   294. The method of embodiment 290, wherein the selected dosage is    non-zero.-   295. The method of embodiment 290, wherein the selected dosage is an    increased dosage.-   296. The method of embodiment 290, wherein the selected dosage is an    reduced dosage.-   297. The method of any of embodiments 196-296, wherein administering    comprises systemic administration, e.g., to the peripheral    circulatory system.-   298. The method of any of embodiments 196-297, wherein administering    comprises local administration, e.g., to the target tissue.-   299. The method of any of embodiments 196-298, comprising    administering a therapeutic compound provided herein such that    therapeutic levels are present for at least 1, 5, 10, 14, or 28    days, e.g, consecutive or non-consequitive days.-   300. The method of any of embodiments 196-299, wherein the subject    is a mammal, e.g., a non-human mammal.-   301. The method of any of embodiments 196-299, wherein the subject    is a human.-   302. A nucleic acid molecule or a plurality of nucleic acid    molecules encoding a therapeutic compound of any of embodiments    1-195.13.-   303. A vector or a plurality of vectors comprising the nucleic acid    molecules of embodiment 302.

304. A cell comprising the nucleic acid molecules of embodiment 302 orthe vector of embodiment 303.

305. A method of making a therapeutic compound comprising culturing acell of embodiment 304 to make the therapeutic compound.

-   306. A method of making a nucleic acid sequence encoding a    therapeutic compound of any of embodiments 1-195.13, comprising

a) providing a vector comprising sequence encoding a targeting moietyand inserting into the vector sequence encoding an effectorbinding/modulating moiety to form a sequence encoding a therapeuticcompound; or

b) providing a vector comprising sequence encoding an effectorbinding/modulating moiety and inserting into the vector sequenceencoding a targeting moiety to form a sequence encoding a therapeuticcompound,

thereby making a sequence encoding a therapeutic compound.

-   307. The method of embodiment 306, wherein the targeting moiety is    selected in response to the need of a subject.-   308. The method of embodiment 306 or 307, wherein the effector    binding/modulating moiety is selected in response to the need of a    subject.-   309. The method of any of embodiments 306 or 307, further comprising    expressing the sequence encoding the therapeutic compound to produce    the therapeutic compound.-   310. The method of any of embodiments 306-309, further comprising    transferring the sequence, or a polypeptide made from the sequence,    to another entity, e.g., a health care provider who will administer    the therapeutic compound to a subject.-   311. A method of treating a subject comprising:

acquiring, e.g., receiving from another entity, a therapeutic compound,or a nucleic acid encoding a therapeutic compound, made by the method ofany of provided herein, but not limited to embodiments 306-310;

administering the therapeutic compound, or a nucleic acid encoding atherapeutic compound to the subject,

thereby treating the subject.

-   312. The method of embodiment 311, further comprising identifying    the therapeutic compound, or nucleic acid encoding a therapeutic    compound to another entity, e.g., the entity that will make the    therapeutic compound, or nucleic acid encoding a therapeutic    compound.-   313. The method of embodiments 311 or 312, further comprising    requesting the therapeutic compound, or nucleic acid encoding a    therapeutic compound from another entity, e.g., the entity that made    the therapeutic compound, or nucleic acid encoding a therapeutic    compound.-   314. The method of any of embodiments 311-333, wherein the subject    has an autoimmune disorder and the therapeutic compound does not    comprise an autoantigenic peptide or polypeptide characteristic of    the autoimmune disorder, e.g., does not comprise a peptide or    polypeptide against which the subject has autoantibodies.

The following examples are illustrative, but not limiting, of thecompounds, compositions and methods described herein. Other suitablemodifications and adaptations known to those skilled in the art arewithin the scope of the following embodiments.

EXAMPLES Example 1: HLA-Targeted PD-1 Agonizing Therapeutic Compounds

Engineering of a HLA-Targeted PD-1-Agonizing Therapeutic

Binding domains specific for HLA-A2 are obtained by cloning the variableregions of the Ig heavy and light chains from the BB7.2 hybridoma (ATCC)and converting into a single-chain Ab (scFv). Activity and specificityof the scFv can be confirmed by assessing binding of BB7.2 to HLA-A2expressing cells in comparison to cells expressing other HLA-A alleles.The minimal PD-L1 residues required for PD-1 binding activity areidentified by systematically evaluating the requirement of amino acids3′ and 5′ of the PD-L1 IgV domain corresponding to amino acids 68-114.Expression constructs are designed and proteins synthesized andpurified, with PD-1 binding activity tested by Biacore. The minimumessential amino acids required for PD-1 binding by the PD-L1 IgV domainare referred to as PD-L1-IgV. To generate a BB7.2 scFv and PD-L1-IgVbi-specific molecule, a DNA fragment is synthesized encoding thebispecific single-chain antibody BB7.2×PD-L1-IgV with the domainarrangement VL_(BB7.2)-VH_(BB7.2)-PD-L1-IgV-IgG4 Fc and cloned into anexpression vector containing a DHFR selection cassette.

Expression vector plasmid DNA is transiently transfected into 293Tcells, and BB7.2×PD-L1-IgV bispecific antibodies are purified fromsupernatants using a protein A/G column. BB7.2×PD-L1-IgV bispecificantibody integrity is assessed by polyacrylamide gel. Binding of theBB7.2 scFv domain to HLA-A2 and PD-L1-IgV domain to PD-1 is assessed byELISA and cell-based FACS assay.

The in vitro function of BB7.2×PD-L1-IgV bispecific antibodies isassessed using mixed lymphocyte reaction (MLR) assay. In a 96-well plateformat, 100,000 irradiated human PBMCs from an HLA-A2⁺ donor arealiquoted per well and used as activators. HLA-A1⁻ responder T cells arethen added together with increasing amounts of BB7.2×PD-L1-IgVbispecific antibody. The ability of responder T cells to proliferateover a period of 72 hours is assessed by BrdU incorporation, and withIFNg and IL2 cytokine production additionally evaluated in theco-culture supernatant as assessed by ELISA. BB7.2×PD-L1-IgV bispecificantibody is found to suppress MLR reaction as demonstrated by inhibitingHLA-A2″ responder T cell proliferation and cytokine production.

The in vivo function of BB7.2×PD-L1-IgV bispecific antibody is assessedusing a murine mouse model of skin allograft tolerance. TheC57BL/6-Tg(HLA-A2.1)1Enge/J (Jackson Laboratories, Bar Harbor Ma.)strain of mouse is crossed with Balb/cJ, with F1 progeny expressing theHLA-A2.1 transgene and serving as allograft donors. C57BL/6J mice areshaved and surgically engrafted with skin removed from euthanizedC57BL/6-Tg(HLA-A2.1)1Enge/J×Balb/cJ F1 mice. At the same time, host micestart receiving intraperitoneal injections of the BB7.2×PD-L1-IgVbispecific antibody engineered to contain a murine IgG1 Fc or BB7.2 onlyor PD-L1-IgV only controls. Skin allograft rejection or acceptance ismonitored over a period of 30 days, wherein hosts were euthanized andlymph node and allograft-resident lymphocyte populations quantified.

Example 2: CD39 and/or CD73 as Effector Domains Creating a PurinergicHalo Surrounding a Cell Type or Tissue of Interest

A catalytically active fragment of CD39 and/or CD73 is fused to atargeting domain. Upon binding and accumulation at the target site, CD39phosphohydrolyzes ATP to AMP. Upon binding and accumulation at thetarget site, CD73 dephosphorylates extracellular AMP to adenosine. Asoluble catalytically active form of CD39 suitable for use herein hasbeen found to circulate in human and murine blood, see, e.g., Yegutkinet al. FASEB J. 2012 September; 26(9):3875-83. A soluble recombinantCD39 fragment is also described in Inhibition of platelet function byrecombinant soluble ecto-ADPase/CD39, Gayle, et al., J Clin Invest. 1998May 1; 101(9): 1851-1859. A suitable CD73 molecule comprises a solubleform of CD73 which can be shed from the membrane of endothelial cells byproteolytic cleavage or hydrolysis of the GPI anchor by shear stresssee, e.g., Reference: Yegutkin G, Bodin P, Burnstock G. Effect of shearstress on the release of soluble ecto-enzymes ATPase and 5′-nucleotidasealong with endogenous ATP from vascular endothelial cells. Br JPharmacol 2000; 129: 921-6.

The local catalysis of ATP to AMP or AMP to adenosine will deplete localenergy stores required for fulminant T effector cell function. Tregfunction should not be impacted by ATP depletion due to their relianceon oxidative phosphorylation for energy needs (which requires less ATP),wherein T memory and other effector cells should be impacted due theirreliance on glycolysis (requiring high ATP usage) for fulminantfunction.

Example 3: Measuring Antibody-Induced PD-1 Signaling

Jurkat cells that stably express 2 constructs, 1) a human PD-1polypeptide fused to a b-galactosidase, which can be referred to as an“Enzyme donor” and 2) a SHP-2 polypeptide fused to a b-galactosidase,which can be referred to as an “Enzyme acceptor.” A PD-1 antibody iscontacted with the cell and when the PD-1 is engaged, SHP-2 is recruitedto PD-1. The enzyme acceptor and enzyme donor form a fully activeb-galactosidase enzyme that can be assayed. This assay can be used toshow activation of PD-1 signaling.

Example 4: Measuring PD-1 Agonism. PD-1 Agonists Inhibit T CellActivation

Without being bound to any particular theory, PD-1 agonism inhibitsanti-CD3-induced T cell activation. Human or mouse cells arepreactivated with PHA (for human T cells) or ConA (for mouse T cells) sothat they express PD-1. The T cells are then “reactivated” with anti-CD3in the presence of anti-PD-1 (or PD-L1) for the PD-1 agonism assay. Tcells that receive a PD-1 agonist signal in the presence of anti-CD3will show decreased activation, relative to anti-CD3 stimulation alone.Activation can be readout by proliferation or cytokine production (IL-2,IFNg, IL-17) and possibly by other markers, such as CD69 activationmarker.

Example 5. Expression and Function of Anti-MAdCAM/Mouse PD-L1 FusionProtein is Not Impacted by Molecular Configuration

A bispecific fusion molecule comprising an anti-mouseMAdCAM Ab/mousePD-L1 molecule was expressed in two orientations. The first orientationconsisted of an anti-mouse MAdCAM IgG with mouse PD-L1 fused at thec-terminus of it's heavy chain. The second orientation consisted ofmouse PD-L1 fused at the N-terminus of an Ig Fc domain, with ac-terminally fused anti-mouse MAdCAM scFv. Both molecules were found tobe well expressed in a mammalioan expression system. It was also foundthat the molecules can bind to their respective binding partners, MAdCAMor PD-1 in both orientations, simultaneously. These results demonstratethat a molecule consisting of an anti-MAdCAM antibody fused to PD-L1,can be expressed in configurations whereby PD-L1 is N or C-terminallyfused to the Fc and retain proper functional binding activity.

Briefly, a pTT5 vector containing the single gene encoding a singlepolypeptide with mouse PD-L1 fused N-terminally of human IgG1 Fc domainand with c-terminal fused anti-MAdCAM scFv MECA-89 was transfected intoHEK293 Expi cells. Alternatively, two plasmids were co-transfected atequimolar ratios. The first plasmid encoded the light chain of MECA-89and the 2nd encoded the full length IgG1 heavy chain of MECA-89 withc-terminally fused mouse PD-L1. After 5-7 days, cell culturesupernatants expressing the molecules were harvested, and clarified bycentrifugation and filtration through a 0.22 um filtration device. Thebi-specific molecules were captured on proA resin. The resin was washedwith PBS pH 7.4 and the captured molecule was eluted using 100 mMGlycine pH 2.5, with neutralization using a tenth volume of 1M Tris pH8.5. The protein was buffer exchanged into PBS pH 7.4, and analyzed bysize exclusion chromatography on a Superdex 200 3.2/300. Analysis of 1μg of purified material by reducing and non-reducing SDS-PAGE on aBis-Tris 4-12% gel was conducted.

Both proteins, regardless of orientation were expressed at over 10 mg/L,and were over 95% monodispersed after purification as shown by sizeexclusion chromatography and reducing/non-reducing SDS-PAGE.Accordingly, this demonstrates the production and activity of dualfunction bispecific molecules with different immunomodulators and tissuetargeting moieties at the N and C terminus of an Fc domain. This alsoshows specifically that a PD-1 agonist and binding partner can beexpressed at the N or C terminus of an Ig Fc domain.

Example 6. A Bispecific Molecule Comprising a PD-1 Agonist ProtoytpeTethered to MAdCAM Can Bind MAdCAM and PD-1 Simultaneously

Briefly, an immunosorbent plate was coated with mouse PD-1 at aconcentration of 1 μg/mL in PBS pH 7.4, 75 ul/well, and incubatedovernight at 4° C. Wells were washed with PBS pH 7.4 containing 0.05%Tween-20 (wash buffer) three times, and then blocked with 200 ul/well 1%BSA in PBS pH 7.4 (block buffer) for two hours at room temperature.After three washes with wash buffer, two bispecific molecules thatcomprises the PD-1 Agonist prototype at either the N-terminus orC-terminus were diluted to 1 nM, 10 nM, and 100 nM in PBS containing 1%BSA and 0.05% Tween-20 (assay buffer). The diluted material was added tothe mouse PD-1 coated plate at 75 ul/well for 1 hour at roomtemperature. After three washes with wash buffer, mouse MAdCAM was addedto the plate at 75 ul/well, at a concentration of 10 nM in assay bufferfor 1 hr at room temperature. After three washes with wash buffer, agoat biotinylated anti-mouse MAdCAM polyclonal antibody, diluted to 0.5μg/mL in assay buffer, was added to the plate at 75 ul/well for 1 hr atroom temperature. After three washes with wash buffer high sensitivitystreptavidin HRP diluted in assay buffer at 1:5000 was added to theplate at 75 ul/well for 15 minutes at room temperature. After threewashes with wash buffer and 1 wash with wash buffer (with no tween-20),the assay was developed with TMB, and stopped with 1N HCL. OD 450 nm wasmeasured. The experiment included appropriate controls for non-specificbinding to the plate/block in the absence of mouse PD-1, as well as noMAdCAM controls, and mono-specific controls, that are unable to form abridge between mouse PD-1 and mouse MAdCAM.

The results demonstrated that at concentrations of 1 nM, 10 nM, and 100nM, both bispecific molecules, are able to simultaneously interact withmouse MAdCAM and mouse PD-L1, whilst the monospecific controls did notcreate a bridging signal. Additionally, there was no binding of anycompound to MAdCAM at any concentration tested, when mouse PD-1 was notpresent on the plate surface, indicating none of the test compounds wereinteracting non-specifically with the plate surface. Thus, these resultsdemonstrate that a bispecific molecule that is targeting binding to bothMAdCAM and PD-1 can successfully bind to both molecules. Although theexperiments were performed with PD-L1 as a substitute for a PD-1antibody, it is expected that the PD-1 antibody will function in asimilar manner.

Example 7. A Bispecific PD-L1 Prototype Molecule Inhibits T Cells in aPD-1 Agonist Assay

A bispecific molecule that mimics a PD-1 agonist antibody was tested todemonstrate that PD-1 agonsim can inhibit T cells. Briefly, 7 week oldfemale C57LB/6 mice were sacrificed and their splenocytes were isolated.The splenocytes were exposed to ConA for 3 days and then exposed toanti-CD3 in the presense or absence of the PD-1 type molecule, which inthis example was a PD-L1 bispecific molecule that was tethered to aplate using anti-human IgG. T cells were then introduced to the PD-L1bispecific molecule. The PD-L1, which mimics a PD-1 antibody were foundto be a T cell agonist and inhibit T cell activation. The sameexperiments were repeated using a PD-L1 bispecific molecule that wasfused with an anti-MAdCAM antibody, which were tethered to a plate byinteracting with a MAdCAM coated plate. The PD-1 agonist mimic, thePD-L1/anti-MAdCAM antibody were found to be effective agonists of T cellactivity. These results demonstrate that a bispecific molecule thatmimics a PD-1 antibody/MAdCAMAb fusion protein can exert functionalinhibitory signaling into primary mouse T cell blasts when the moleculeis captured via the MAdCAM antibody component at the end of themolecule.

Example 8: A bispecific PD-1 Prototype Molecule With a Different TissueTether Can Inhibit T Cells in a PD-1 Agonist Assay

A fusion molecule of a PD-L1 was used as a substitute for a PD-1antibody and linked to a Class I H-2Kk antibody. The MHC Class I H-2Kktethered PD-L1 molecule had functional binding, similar to the datadescribed in Examples 6 and 7. Briefly, splenocytes from C57Bl/6 micewere stimulated with Concanavalin A (ConA) and IL-2 for 3 days. Plateswere coated with anti-CD3 (2C11) overnight at 4 C, washed. Plates werecoated with anti-human IgG for 3 hrs at 37 C and washed. Mono-specificanti-H-2Kk (16-3-22) or bi-specific anti-H-2Kk: mPD-L1 were added andincubated for 3 hr at 37 C and washed. All test articles contained ahuman IgG1-Fc portion. PBS (No Tx) was added to determine the assaybackground. ConA blasts were washed 2 times, added to the plate andincubated at 37 C. Supernatants were removed after 24 hrs. IFNg levelswere determined by MSD. After 48 hrs, cell viability/metabolism wasanalyzed by Cell Titer-glo. When captured via the IgG Fc domain, an MHCClass I tethered PD-L1 bispecific can attenuate T cell activation in amouse PD-1 agonism assay. Therefore, this example demonstrates that adifferent bispecific prototype molecule can exert functional inhibitorysignaling into primary mouse T cell blasts—when the molecule is capturedvia a different tissue tether—in this case a mouse antibody to MHC ClassI H-2Kk. Accordingly, this data demonstrates that the tethering is notspecific to MAdCAM and is possible with other molecules that can act astargeting moieties as provided herein.

Example 9. PD-1 Agonists Can Induce Signaling in Jurkat Cells

Jurkat cells expressing both human PD-1 fused to a beta-galactosidaseenzyme donor and SHP-2 fused to a beta-galactosidase enzyme acceptor areadded to test conditions in a plate and incubated for 2 hrs. AgonistPD-1 antibodies induce signaling and SHP-2 recruitment, enzymecomplementation and formation of an active beta-galactosidase enzyme.Beta-galactosidase substrate was added and chemiluminescence can bemeasured on a standard luminescence plate reader. Agonism is measured bychemiluminescence, where the more chemiluminescence that is measuredindicates the greater agonism.

Agonism of a PD-1/MAdCAM bi-specific molecule was measured in thisassay. C110 (UCB) and CC-90006 (Celgene/Anaptys) were used as PD-1agonist antibodies. Both are active and exhibit PD-1 agonism infunctional assay in Ig-capture assay format. Briefly, plates were coatedwith anti-human IgG for overnight at 4 C and washed. Anti-tetanus toxin(TT) or benchmark agonist anti-PD-1 monoclonal antibodies, C110 orCC-90006 were added and incubated for 1 hr at 37 C and washed. All testarticles contained a human IgG1-Fc. Media (No Tx) was added to determinethe assay background. Plates were washed 3 times. Jurkat cellsexpressing both human PD-1 fused to a b-galactosidase enzyme donor andSHP-2 fused to a b-galactosidase enzyme acceptor were added andincubated for 2 hrs. Agonist PD-1 antibodies induce signaling and SHP-2recruitment, enzyme complementation and formation of an activeb-galactosidase enzyme. B-galactosidase substrate was added andchemiluminescence was measured on a standard luminescence plate reader.The two human PD-1 agonist antibodies (C110 and CC-90006) bind andinduce signaling (a surrogate for agonism) in the modified Jurkatreporter assay. Thus, this assay is a functional PD-1 agonism assay.C110:MECA89 (MECA89 is a known MAdCAM antibody) is a novel bispecificmolecule created by fusing MAdCAM antibody, MECA89[scFv], to C-terminusof the heavy chain of C110. This fusion protein was found to be activeand exhibit PD-1 agonism in functional assay when captured via IgG Fcdomain, as was C110 only protein. However, only C110:MECA89 is active infunctional assay format using MAdCAM protein as capture (themonospecific components do not signal).

Briefly, plates were coated with either anti-human IgG or recombinantmMAdCAM-1 overnight at 4 C and washed. Mono-specific Anti-tetanus toxin(TT), anti-MAdCAM-1 (MECA89) or agonist anti-PD-1 (C110) or bi-specificC110:MECA89 were added and incubated for 1 hr at 37 C and washed. Alltest articles contained a human IgG1-Fc portion. PBS (No Tx) was addedto determine the assay background. Plates were washed 2 times. Jurkatcells expressing both human PD-1 fused to a b-galactosidase enzyme donorand SHP-2 fused to a b-galactosidase enzyme acceptor were added andincubated for 2 hrs. Agonist PD-1 antibodies induce signaling and SHP-2recruitment, enzyme complementation and formation of an activeb-galactosidase enzyme. B-galactosidase substrate was added andchemiluminescence was measured on a standard luminescence plate reader.Results: Both C110, and the MAdCAM-tethered C110 bispecific moleculescan induce PD-1 signaling in the Jurkat reporter assay when the plate iscoated with an anti-IgG Fc capture, but only the MAdCAM-tetheredbispecific can induce PD-1 signaling in the reporter assay when theplate is coated with recombinant MAdCAM protein. These resultsdemonstrate that the molecule tethered with MAdCAM and contains a PD-1agonist antibody are functional, which is similar to the results shownwith the PD-L1 as the PD-1 agonist surrogate.

Example 10: Generation of PD-1 Agonist Antibodies

PD-1 deficient mice immunized with mouse PD-1 under conditions togenerate an immune response against PD-1. 54 hybridomas were generatedand identified that bind mouse PD-1. The antibodies produced by thedifferent hybridomas were analyzed for T cell agonism according to themethods described in Examples 4 and 6. Out of the 54 hybridomas at least6 were identified as PD-1 agonists. The antibodies were also tested forbinding on PD-1 and were found to bind at the same site as the PD-L1binding site.

Briefly, binding to the PD-L1 binding site was determined using thefollowing assay. Immunosorbent plates were coated overnight with 75 μLof recombinant mouse PD-L1-Fc (2 μg/mL) in 1×PBS, pH 7.4. Plates werethen washed 3× with 1×PBS and blocked for 2 hours at room temperaturewith 1×PBS supplemented with 1% BSA. Recombinant mouse PD-1-Fc (1 nM)was incubated with 100 nM of the indicated anti-mouse PD-1 antibody in1×PBS supplemented with 1% BSA and 0.05% Tween20 (Assay Buffer) for 1hour at room temperature, shaking. After blocking, plates were washed 3×with 1×PBS supplemented with 0.05% Tween20 PB ST and the antibody-PD-1conjugates were incubated with plate-bound mouse PD-L1. After washingaway unbound PD-1 with PBST, plates were incubated with 75 μL ofbiotinylated, polyclonal anti-PD-1 antibody (0.5 μg/mL) in assay buffer,followed by amplification with 1:5000 streptavidin HRP also diluted inassay buffer. Plates were washed three times with PB ST followed bythree washes with 1×PBS before addition of 100 μL TMB followed by 100 μL1M HCl to stop the developing. Absorbance read at 450 nm and normalizedto binding of PD-1 to PD-L1 in the absence of antibody. The resultsshowed that the active antibodies bind to the PD-L1 binding site. Theinactive antibodies did not bind to the PD-L1 binding site. Therefore,this example demonstrates the ability to produce anti-PD-1 antibodiesthat are agonists, in addition to the previously identified PD-1 agonistantibodies described herein.

Example 11: Tethered Anti-PD-1 Antibodies Acts as PD-1 Agonists

A human antibody scFv phage library was panned against recombinanthuman, mouse, and cyno PD-1 proteins across iterative selection roundsto enrich for antibody clones that recognize all three aforementionedspecies orthologues of PD-1. The scFv clones were configured innt-VH-Linker-VL-ct format and fused to the M13 phage surface via thepIII coat protein. After selections, clonal scFvs were screened forbinding to human, mouse, and cyno PD-1 expressed on the cell surface ofCHO cells. Clones that were found to be cross reactive to all three cellsurface expressed PD-1 species orthologues were converted using standardmolecular biology techniques, into a human IgG1 format whereby eachmolecule was comprised of four polypeptide chains in total (2 heavy, and2 light chains). The two light chains were identical to each other andthe two heavy chains were identical to each other as provided.

The two identical heavy chains homodimerize and the two identical lightchains pair with each heavy chain to form an intact human IgG1. The Fcdomain contains the L234A, L235A, and G237A mutations to ablate FcγRinteractions. The converted human IgG1 anti-PD-1 antibodies weretransfected and expressed in HEK293 Expi cells, and purified by proteinA chromatography. The protein concentration was determined using ananodrop spectrophotometer in conjunction with antibody specificextinction coefficients. Antibodies were formulated in PBS pH 7.4.

The anti-PD-1 antibodies were next tested in the Jurkat assay describedherein for agonist activity. Briefly, tissue culture plates were coatedwith anti-IgG or left uncoated. For captured format, test articles orcontrols were added to the anti-IgG coated wells at 100 nM, 25 nM or12.5 nM and incubated for 3 hrs at 37 C. Plates were washed and JurkatPD-1 cells were added. For the soluble format, soluble test articles orcontrols were added to wells at 100 nM, 25 nM or 12.5 nM alreadycontaining Jurkat PD1 cells. Luminescence was measured in a platereader. The results demonstrated that nine of the twelve human/mousecross-reactive PD-1 antibodies showed dose-dependent activity in theJurkat assay when the anti-PD-1 antibodies were captured via anti-IgG,but not in the soluble format. This data demonstrates that the anti-PD-1antibody can act as an agonist when tethered to its target by atargeting moiety.

In conclusion, without being bound to any particular theory, the datapresented herein demonstrate that a PD-1 Agonist/MAdCAM bi-specificmolecule can bind to both MAdCAM and PD-1 and also agonize T cellactivity. Thus, the molecules can be used to treat the variousconditions provided herein and provide for localized and/or tissuespecific immunomodulation and the down regulation of a T-Cell response.

Example 12: Generation of IL-2 Muteins

A pTT5 vector containing the single gene encoding the human IL-2Mpolypeptide fused N-terminally (SEQ ID NO: 57) or C-terminally (SEQ IDNO: 58) to human IgG1 Fc domain was transfected into HEK293 Expi cells.After 5-7 days, cell culture supernatants expressing IL-2Ms wereharvested, and clarified by centrifugation and filtration through a 0.22um filtration device. IL-2Ms were captured on proA resin. The resin waswashed with PBS pH 7.4 and the captured protein was eluted using 0.25%acetic acid pH 3.5, with neutralization using a tenth volume of 1M TrispH 8.0. The protein was buffer exchanged into 30 mM HEPES 150 mM NaCl pH7, and analyzed by size exclusion chromatography on a Superdex 2003.2/300 column. Analysis of 5 ug of purified material by reducing andnon-reducing SDS-PAGE on a Bis-Tris 4-12% gel was conducted. The IL-2Mswere expressed at over 10 mg/L, and were over 95% monodispersed afterpurification as shown by size exclusion chromatography andreducing/non-reducing SDS-PAGE.

Example 13: IL-2M Molecules Can Bind CD25

An immunosorbent plate was coated with CD25 at a concentration of 0.5μg/mL in PBS pH 7.4, 75 ul/well, and incubated overnight at 4° C. Wellswere washed with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer)three times, and then blocked with 200 ul/well 1% BSA in PBS pH 7.4(block buffer) for two hours at room temperature. After three washeswith wash buffer IL-2M molecules of Example 12 were diluted toeleven-two fold serial dilution in PBS containing 1% BSA and 0.05%Tween-20 (assay buffer) with 2 nM being the highest concentration. Thediluted material was added to the CD25 coated plate at 75 ul/well for 1hour at room temperature. After three washes with wash buffer, a goatbiotinylated anti-IL-2 polyclonal antibody, diluted to 0.05 μg/mL inassay buffer, was added to the plate at 75 ul/well for 1 hr at roomtemperature. After three washes with wash buffer high sensitivitystreptavidin HRP diluted in assay buffer at 1:5000 was added to theplate at 75 ul/well for 15 minutes at room temperature. After threewashes with wash buffer and 1 wash with wash buffer (with no tween-20),the assay was developed with TMB, and stopped with 1N HCL. OD 450 nm wasmeasured. The experiment included appropriate controls for non-specificbinding of IL-2M molecules to the plate/block in the absence of CD25 anda negative control molecule that is unable to bind CD25.

The results indicate that at concentrations of 2 nM-1.9 pM, IL-2Mmolecules are able to bind CD25 with sub nanomolar EC50s. Additionally,there was no detection of any compound at any concentration tested, whenCD25 was not present on the plate surface, indicating none of the testcompounds were interacting non-specifically with the plate surface (datanot shown).

Example 14: In Vitro P-STAT5 Assay to Determine Potency and Selectivityof IL-2M Molecules

Peripheral blood mononuclear cells (PBMCs) were prepared usingFICOLL-PAQUE Premium and Sepmate tubes from freshly isolated heparinizedhuman whole blood. PBMCs were cultured in 10% fetal bovine serum RPMImedium in the presence of wild-type IL-2 or IL-2M of Example 12 for 20minutes and then fixed for 10 minutes with BD Cytofix. Fixed cells weresequentially permeabilized with BD Perm III and then BioLegend FOXP3permeabilization buffer. After blocking with human serum for 10 minutes,cells were stained for 30 minutes with antibodies for phospho-STAT5FITC, CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 and then acquired on anAttune NXT with plate reader. The IL-2M of Example 12 potently andselectively induces STAT5 phosphorylation in Tregs but not Teffs.

Example 15: Methods for Generation of Bispecific MAdCAM-Tethered IL-2MMolecules

A pTT5 vector containing the single gene encoding the single B0001polypeptide comprising an IL-2 mutein with a N88D, V69A, and Q74Pmutations fused to a Fc protein with the LALA mutations as provided forherein with a GGGGS(×3) linker and scFV antibody that binds to MAdCAM ora similar molecule but with a GGGGS(×4) linker B0002 with human IL-2Mfused N-terminally of human IgG1 Fc domain and with c-terminal fusedanti-mMAdCAM scFv MECA-89 was transfected into HEK293 Expi cells. ForB0003, two plasmids were co-transfected at equimolar ratios. The firstplasmid encoded the light chain of MECA-89 and the 2nd encoded the fulllength IgG1 heavy chain of MECA-89 with c-terminally fused human IL-2M.After 5-7 days, cell culture supernatants expressing B0001, B0002, andB0003 were harvested, and clarified by centrifugation and filtrationthrough a 0.22 um filtration device. B0001, B0002, and B0003 werecaptured on proA resin. The resin was washed with PBS pH 7.4 and thecaptured protein was eluted using 0.25% acetic acid pH 3.5, withneutralization using a tenth volume of 1M Tris pH 8.0. The protein wasbuffer exchanged into 30 mM HEPES 150 mM NaCl pH 7, and analyzed by sizeexclusion chromatography on a Superdex 200 3.2/300. Analysis of 1 μg ofpurified material by reducing and non-reducing SDS-PAGE on a Bis-Tris4-12% gel was conducted.

B0001, B0002, and B0003 were expressed at over 8 mg/L, and were over 95%monodispersed after purification as shown by size exclusionchromatography and reducing/non-reducing SDS-PAGE. This experiment showsthat dual function bispecific molecules with immunomodulators at eitherthe N or C terminus can be produced and the position of the IL-2Mprotein (either at the N or C terminus) did not significantly alterexpression and therefore, either format can be used.

Example 16: Bispecific MAdCAM-Tethered IL-2M Molecules Can Bind MAdCAMand CD25 Simultaneously

An immunosorbent plate was coated with recombinant mouse MAdCAM-1 at aconcentration of 1 μg/mL in PBS pH 7.4, 75 ul/well, and incubatedovernight at 4° C. Wells were washed with PBS pH 7.4 containing 0.05%Tween-20 (wash buffer) three times, and then blocked with 200 ul/well 1%BSA in PBS pH 7.4 (block buffer) for two hours at room temperature.After three washes with wash buffer, B0001, B0002, B0003 were diluted to1 nM, 10 nM, and 100 nM in PBS containing 1% BSA and 0.05% Tween-20(assay buffer). The diluted material was added to the mouse MAdCAM-1coated plate at 75 ul/well for 1 hour at room temperature. After threewashes with wash buffer, human CD25 was added to the plate at 75ul/well, at a concentration of 10 nM in assay buffer for 1 hr at roomtemperature. After three washes with wash buffer, a goat biotinylatedanti-human CD25 polyclonal antibody, diluted to 0.4 μg/mL in assaybuffer, was added to the plate at 75 ul/well for 1 hr at roomtemperature. After three washes with wash buffer high sensitivitystreptavidin HRP diluted in assay buffer at 1:5000 was added to theplate at 75 ul/well for 15 minutes at room temperature. After threewashes with wash buffer and 1 wash with wash buffer (with no tween-20),the assay was developed with TMB, and stopped with 1N HCL. OD 450 nm wasmeasured. The experiment included appropriate controls for non-specificbinding of the proteins of Example 15 to the plate/block in the absenceof mouse MAdCAM-1, as well as no CD25 controls, and mono-specificcontrols, that are unable to form a bridge between human CD25 and mouseMAdCAM.

It was found that at concentrations of 1 nM, 10 nM, and 100 nM, thebi-specific molecules of Example 15 were able to simultaneously interactwith mouse MAdCAM and human CD25, whilst the monospecific controls, didnot create a bridging signal. Additionally, there was no binding of anycompound to CD25 at any concentration tested, when mouse MAdCAM-1 wasnot present on the plate surface, indicating none of the test compoundswere interacting non-specifically with the plate surface. These resultsdemonstrate that the bispecific molecules can bind both MAdCAM and CD25simultaneously in a functional binding assay, such as an ELISA.

Example 17: In Vitro P-STAT5 Assay Demonstrating Activity andSelectivity of Bispecific MAdCAM-Tethered IL-2M When In Solution or WhenTethered

Recombinant mouse MAdCAM was coated onto wells of a 96 well high bindingplate (Corning) overnight. After washing 2 times with PBS, the plate wasblocked for 1 hour with 10% FBS RPMI media. A MAdCAM-tethered IL-2Mbispecific of Example 15 or untethered IL-2M control (such as thoseprepared in Example 12) were captured for 1 hour. After washing 2 timeswith PBS, freshly-isolated human PBMCs were stimulated for 60 minuteswith captured IL-2M or for comparison IL-2M in solution. Cells were thenfixed for 10 minutes with BD Cytofix, permeabilized sequentially with BDPerm III and BioLegend FOXP3 permeabilization buffer, blocked with humanserum and stained for 30 minutes with antibodies against phospho-STAT5FITC (CST), CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 (BD) and acquiredon an Attune NXT with plate loader. In solution, both molecules havecomparable activity and selectivity on Treg versus Teff. Plates coatedwith mouse MAdCAM were able to capture the bi-specific molecule ofExample 15 and the captured/immobilized bi-specific molecule was stillable to selectively activate T_(regs) over T_(effs). This exampledemonstrates that MAdCAM-tethered IL-2M molecules can retain biologicalactivity and selectivity when in solution or when captured/immobilized.

Example 18: Immunogenicity of IL-2 Muteins

IL-2 Mutein sequences were analyzed using the NetMHCIIPan 3.2 software,which can be found at www “dot” cbs “dot” dtu “dot”dk/services/NetMHCIIpan/. Artificial neural networks were used todetermine peptide affinity to MHC class II alleles. In that analysis,9-residue peptides with potentially direct interaction with the MHCclass II molecules were recognized as binding cores. Residues adjacentto binding cores, with potential to influence the binding indirectly,were also examined as masking residues. Peptides comprising both thebinding cores and masking residues were marked as strong binders whentheir predicted K_(D) to the MHC class II molecule was lower than 50 nM.Strong binders have a greater chance of introducing T cellimmunogenicity.

A total of 9 MHCII alleles that are highly represented in North Americaand Europe were included in the in silico analysis. The panel of IL-2M(IL-2 mutein) molecules tested included the IL-2 Muteins with L53I,L56I, L80I, or L118I mutations. Only MHCII alleles DRB1_1101, DRB1_1501,DRB1_0701, and DRB1_0101 yielded hits with any of the moleculesassessed. The peptide hits for DRB_1501 were identical between allconstructs tested including wild-type IL-2 with the C125S mutation. Theaddition of L80I removes 1 T cell epitope for DRB1-0101[ALNLAPSKNFHLRPR] and modestly reduces the affinity of two other T cellepitopes [EEALNLAPSKNFHLR and EALNLAPSKNFHLRP]. For MHCII alleleDRB1-0701, L80I removes 1 T cell epitope [EEALNLAPSKNFHLR]. Therefore,the data demonstrates that a IL-2 mutein comprising the L80I mutationshould be less immunogenic, which is a surprising and unexpected resultfrom the in silico analysis.

Example 19: Generation of Additional IL-2 Muteins

A pTT5 vector containing the single gene encoding the single IL-2M (IL-2mutein) of SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56(and IL-2M control; SEQ ID NO: 50) polypeptide with human IL-2M fusedN-terminally of human IgG1 Fc domain was transfected into HEK293 Expicells. After 5-7 days, cell culture supernatants expressing SEQ ID NO:53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 (and IL-2M control; SEQID NO: 50) were harvested, and clarified by centrifugation andfiltration through a 0.22 um filtration device. SEQ ID NO: 53, SEQ IDNO: 54, SEQ ID NO: 55, SEQ ID NO: 56 (and IL-2M control; SEQ ID NO: 50)were captured on proA resin. The resin was washed with PBS pH 7.4 andthe captured protein was eluted using 0.25% acetic acid pH 3.5, withneutralization using a tenth volume of 1M Tris pH 8.0. The protein wasbuffer exchanged into 30 mM HEPES 150 mM NaCl pH 7, and analyzed by sizeexclusion chromatography on a Superdex 200 3.2/300 column. Analysis of 5ug of purified material by reducing and non-reducing SDS-PAGE on aBis-Tris 4-12% gel was conducted.

IL-2Ms SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 (andIL-2M control; SEQ ID NO: 50) expressed at over 45 mg/L, and were over95% monodispersed after purification as shown by size exclusionchromatography and reducing/non-reducing SDS-PAGE.

Example 20: IL-2Ms of Example 19 can Bind CD25

An immunosorbent plate was coated with CD25 at a concentration of 0.5μg/mL in PBS pH 7.4, 75 ul/well, and incubated overnight at 4° C. Wellswere washed with PBS pH 7.4 containing 0.05% Tween-20 (wash buffer)three times, and then blocked with 200 ul/well 1% BSA in PBS pH 7.4(block buffer) for two hours at room temperature. After three washeswith wash buffer IL-2Ms SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQID NO: 56 were diluted to eleven-two fold serial dilution in PBScontaining 1% BSA and 0.05% Tween-20 (assay buffer) with 2 nM being thehighest concentration. The diluted material was added to the CD25 coatedplate at 75 ul/well for 1 hour at room temperature. After three washeswith wash buffer, a goat biotinylated anti-IL-2 polyclonal antibody,diluted to 0.05 μg/mL in assay buffer, was added to the plate at 75ul/well for 1 hr at room temperature. After three washes with washbuffer high sensitivity streptavidin HRP diluted in assay buffer at1:5000 was added to the plate at 75 ul/well for 15 minutes at roomtemperature. After three washes with wash buffer and 1 wash with washbuffer (with no tween-20), the assay was developed with TMB, and stoppedwith 1N HCL. OD 450 nm was measured. The experiment included appropriatecontrols for non-specific binding of the molecules to the plate/block inthe absence of CD25. The results indicate that at concentrations of 2nM-1.9 pM, the muteins of Example 19 were able to bind CD25 with subnanomolar EC50s. Additionally, there was no detection of any compound atany concentration tested, when CD25 was not present on the platesurface, indicating none of the test compounds were interactingnon-specifically with the plate surface. Thus, the muteins of Example 19can bind to CD25.

Example 21: IL-2 Muteins of Example 19 are Potent and Selective

Peripheral blood mononuclear cells (PBMCs) were prepared usingFICOLL-PAQUE Premium and Sepmate tubes from freshly isolated heparinizedhuman whole blood. PBMCs were cultured in 10% fetal bovine serum RPMImedium in the presence of wild-type IL-2 or the muteins of Example 19for 20 minutes and then fixed for 10 minutes with BD Cytofix. Fixedcells were sequentially permeabilized with BD Perm III and thenBioLegend FOXP3 permeabilization buffer. After blocking with human serumfor 10 minutes, cells were stained for 30 minutes with antibodies forphospho-STAT5 FITC (CST), CD25 PE, FOXP3 AF647 and CD4 PerCP Cy5.5 (allBD) and then acquired on an Attune NXT with plate reader. The IL-2muteins of Example 19 were found to be potent and have selectivityagainst Treg versus Teff. The mutein comprising the L118I mutation wasfound to have increased activity and selectivity as compared to theother muteins.

Example 22: IL-2 Muteins Expand Tregs in Humanized Mice

NSG mice humanized with human CD34+ hematopoietic stem cells werepurchased from Jackson Labs. On days 0 and 7, the mice were dosedsubcutaneously with 1 μg IL-2 Mutein (SEQ ID NO: 50) or other IL-2muteins SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 56.On Day 7, mice were euthanized and whole blood and spleens werecollected. Whole blood was aliquoted into a 96 well deep well plate andfixed for 10 minutes using BD Fix Lyse. Splenocytes were isolated using70 um filters (BD) and red blood cells were lysed using RBC lysis bufferfrom BioLegend. After washing with 2% fetal bovine serum PBS,splenocytes were labeled with near infrared live dead stain (Invitrogen)for 20 minutes and then fixed for 20 minutes using BioLegend fixationbuffer. Both whole blood cells and splenocytes were then permeabilizedusing BioLegend FOXP3 permeabilization buffer, blocked with human serumand stained for 30 minutes with antibodies against human CD8a FITC (BL),human CD25 PE (BD), human FOXP3 AF647 (BD) CD4 PerCP Cy5.5 (BD), humanSiglec-8 PE Cy7 (BL), human CD3 BV421 (BL), human CD45 BV605 (BL), humanCD56 BV785 (BL) and mouse CD45 (BV711) and acquired on an Attune NXTwith plate loader.

Compared to vehicle control, IL-2Ms SEQ ID NO: 54 and SEQ ID NO: 56selectively induced Tregs in mouse spleens and whole blood (p<0.0005 byANOVA with Dunn's Multiple Comparison Test). The other IL-2Ms alsoincreased the frequency of Tregs, though these changes compared to thevehicle group were not statistically significant. There were nosignificant changes in the frequencies of CD56pos NK cells, CD3pos Tcells, CD8pos cytotoxic T lymphocytes, CD4pos helper T cells orCD25lo/FOXP3neg T effectors in mice dosed with SEQ ID NO: 54 and SEQ IDNO: 56. These results demonstrate that the IL-2 muteins increase thefrequency of regulatory T cells.

Example 23: Generation of Bispecific mMAdCAM-Tethered IL-2M Molecule

A bispecific MAdCAM-IL-2 mutein was produced, with the antibody beingthe heavy and light chains of MECA89. This was produced using twoplasmids encoding both heavy and light chains were co-transfected atequimolar ratios. The first plasmid encoded the light chain of MECA-89and the second encoded the full length IgG1 heavy chain of MECA-89 withC-terminally fused to a human IL-2M comprising the L118I mutation. After3-5 days, cell culture supernatants expressing the bispecific wereharvested, and clarified by centrifugation and filtration through a 0.22um filtration device. The bispecific was captured on proA resin. Theresin was washed with PBS pH 7.4 and the captured protein was elutedusing 0.25% acetic acid pH 3.5, with neutralization using a tenth volumeof 1M Tris pH 8.0. The protein was buffer exchanged into 30 mM HEPES 150mM NaCl pH 7, and analyzed by size exclusion chromatography on anAdvanceBio SEC column. Analysis of 1 μg of purified material by reducingand non-reducing SDS-PAGE on a Bis-Tris 4-12% gel was conducted.

The bispecific molecule expressed at 17 mg/L, and was over 95%monodispersed after purification as shown by size exclusionchromatography and reducing/non-reducing SDS-PAGE. These resultsdemonstrate that it was able to produce dual function bispecificmolecules with immunomodulators at the C terminus.

Example 24: Generation of MAdCAM Antibodies

A human antibody scFv phage library was panned against recombinanthuman, mouse, and cyno MAdCAM proteins across iterative selection roundsto enrich for antibody clones that recognize all three aforementionedspecies orthologues of MAdCAM. The scFv clones were configured innt-VH-Linker-VL-ct format and fused to the M13 phage surface via thepIII coat protein. After selections, clonal scFvs were screened by ELISAfor binding to human, mouse, and cyno MAdCAM expressed on the cellsurface of CHO cells. Clones that were found to be cross reactive to allthree cell surface expressed MAdCAM species orthologues were convertedusing standard molecular biology techniques or gene synthesis, into ahuman IgG1 format whereby each molecule was comprised of fourpolypeptide chains in total (2 heavy, and 2 light chains). The two lightchains were identical to each other and the two heavy chains wereidentical to each other. The two identical heavy chains (1 and 2)homodimerize and the two identical light chains (3 and 4) pair with eachheavy chain to form an intact human IgG1. The Fc domain contains theL234A, L235A, and G237A mutations to ablate FcγR interactions. Theformat can be illustrated as follows:

Chain 1: nt-VH1-CH1-CH2-CH3-ct

Chain 2: nt-VH1-CH1-CH2-CH3-ct

Chain 3: nt-VK1-CK-ct

Chain 4: nt-VK1-CK-ct

In addition, MAdCAM scFvs were also converted using standard molecularbiology techniques (such as Gibson Cloning procedure) or gene synthesisinto a bispecific format whereby an IL-2M was situated at the c-terminusof the IgG heavy chain of the MAdCAM antibody, as outlined below:

Chain 1: nt-VH1-CH1-CH2-CH3-ct-Linker-IL-2M

Chain 2: nt-VH1-CH1-CH2-CH3-ct-Linker-IL-2M

Chain 3: nt-VK1-CK-ct

Chain 4: nt-VK1-CK-ct

An ELISA was used to analyze binding of anti-MAdCAM scFvs to captured orplate bound human, cyno, and mouse MAdCAM. Biotinylated human and cynoMAdCAM were captured on a streptavidin coated plate, and mouse MAdCAM-Fccoated directly onto an immunosorbent plate. After a blocking step, theplates were washed and scFv in crude periplasmic lysate was applied tothe plate surface. scFv binding was detected using an anti-VS HRPconjugate. The assay was developed with TMB substrate and stopped withacid. The absorbance at 450 nm was measured. Appropriate wash steps wereapplied between each step of the ELISA. Human versus cyno and humanversus mouse were evaluated. The scFv's were also analyzed using surfaceplasmon resonance technology. After being captured on a biosensorsurface via the V5 tag, soluble monomeric human MAdCAM was titrated andboth binding and dissociation measured and fit to a 1:1 binding modelallowing the derivation of on and off-rates.

The results measured indicate that the majority of clones tested havehuman and cyno MAdCAM binding cross reactivity and a small panel haveadditional cross reactivity to mouse MAdCAM. Biosensor experimentsdemonstrated that the clones exhibited a range of binding on andoff-rates against human MAdCAM with k_(a) values ranging from 10³ 1/Msthrough 10⁷ 1/Ms and k_(d) values ranging 10⁻¹ through 10⁻⁴ 1/s. Certainclones have an off-rate slower than 2×10e2 1/s. Thus, MadCAM antibodieswere generated and can be used in a bi-specific format.

Example 25: Generation of Bispecific Human MAdCAM-Tethered IL-2Ms ofExample 19

Two plasmids each were co-transfected at equimolar ratios. The firstplasmid in each case encoded the light chain of Hu.MAdCAM and the secondencoded the full length IgG1 heavy chain of Hu.MAdCAM with aC-terminally fused human IL-2M comprising the L118I mutation asillustrated in the Table of MAdCAM-IL-2 Mutein Bispecific Compoundsprovided herein. After 3-5 days, cell culture supernatants expressingthe Hu.MAdCAM-IL-2M bispecifics was harvested, and clarified bycentrifugation and filtration through a 0.22 um filtration device. TheHu.MAdCAM-IL-2M bispecifics were captured on proA resin. The resin waswashed with PBS pH 7.4 and the captured proteins were eluted using 0.25%acetic acid pH 3.5, with neutralization using a tenth volume of 1M TrispH 8.0. The proteins were buffer exchanged into 30 mM HEPES 150 mM NaClpH 7, and analyzed by size exclusion chromatography on an AdvanceBio SECcolumn. Analysis of 1 μg of purified material by reducing andnon-reducing SDS-PAGE on a Bis-Tris 4-12% gel was conducted. TheHu.MAdCAM-IL-2M bispecifics expressed at over 10 mg/L, and was over 95%monodispersed after purification as shown by size exclusionchromatography and reducing/non-reducing SDS-PAGE. Thus, these resultsdemonstrate that fully human dual function bispecific molecules withimmunomodulators at the C terminus can be produced.

Example 26: Durability of Signaling Induced by IL-2 Muteins

Peripheral blood mononuclear cells (PBMCs) were prepared usingFICOLL-PAQUE Premium and Sepmate tubes from freshly isolated heparinizedhuman whole blood. PBMCs were cultured in 10% fetal bovine serum RPMImedium in the presence of IL-2Ms for 60 minutes. Cells were then wash 3times and incubated for an additional 3 hours. Cells were then fixed for10 minutes with BD Cytofix. Fixed cells were sequentially permeabilizedwith BD Perm III and then BioLegend FOXP3 permeabilization buffer. Afterblocking with human serum for 10 minutes, cells were stained for 30minutes with antibodies for phospho-STAT5 FITC, CD25 PE, FOXP3 AF647 andCD4 PerCP Cy5.5 and then acquired on an Attune NXT with plate reader.All four IL-2 muteins of Example 19 induced durable signaling in Tregbut not in Teff as compared to the control. An IL-2 mutein of SEQ ID NO:56 is superior to an IL-2 mutein of SEQ ID NO: 55, SEQ ID NO: 54 or SEQID NO: 53. These results demonstrate that the IL-2 can induce durableand selective signaling in Treg which should lead to greater Tregexpansion in vivo and permit less frequent dosing to achieve Tregexpansion.

Example 27: In Vitro P-STAT5 Assay Demonstrates Activity and Selectivityof Bispecific Hu.MAdCAM-Tethered IL-2 Muteins When in Solution or WhenTethered

Recombinant human MAdCAM was coated onto wells of a 96 well high bindingplate (Corning) overnight. After washing 2 times with PBS, the plate wasblocked for 1 hour with 10% FBS RPMI media. MAdCAM-tethered IL-2M muteinbi-specifics or untethered IL-2M control were captured for 1 hour. Afterwashing 2 times with PBS, freshly-isolated human PBMCs were stimulatedfor 60 minutes with captured IL-2MM or for comparison IL-2MM insolution. Cells were then fixed for 10 minutes with BD Cytofix,permeabilized sequentially with BD Perm III and BioLegend FOXP3permeabilization buffer, blocked with human serum and stained for 30minutes with antibodies against phospho-STAT5 FITC (CST), CD25 PE, FOXP3AF647 and CD4 PerCP Cy5.5 (BD) and acquired on an Attune NXT with plateloader. Results: In solution, IL-2M bi-specifics tethered to humanMAdCAM and the control have comparable activity and selectivity on Tregversus Teff. Plates coated with MAdCAM were able to capturebi-specifics, and the captured/immobilized bi-specifics were still ableto selectively activate Tregs over Teffs. This example demonstrates thatIL-2MM bi-specifics targeting human MAdCAM can retain biologicalactivity and selectivity when in solution or when captured/immobilized.

The examples provided for herein demonstrate the surprising andunexpected result that a bispecific molecule comprising a MAdCAMantibody and a IL-2 mutein can function to selectively and potentlyactivate Tregs over Teffs, which demonstrates that the molecules can beused to treat or ameliorate the conditions described herein. Theexamples also demonstrate that the IL-2 mutein can function toselectively and potently activate Tregs over Teffs when used alone (orlinked to a Fc protein) as provided for herein.

The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety. While various embodiments have been disclosed withreference to specific aspects, it is apparent that other aspects andvariations of these embodiments may be devised by others skilled in theart without departing from the true spirit and scope of the embodiments.The appended claims are intended to be construed to include all suchaspects and equivalent variations.

What is claimed is:
 1. A method of increasing T regulatory cellproliferation in a subject, the method comprising administering to thesubject a pharmaceutical composition comprising a polypeptide comprisingan antibody that binds to MAdCAM on the cell surface and an IL-2 muteinpolypeptide (IL-2 mutein), wherein the IL-2 mutein comprises the aminoacid sequence of SEQ ID NO: 6 comprising one or more mutations of E15Q,H16N, Q22E, D84N, E95Q, Q126E, N29S, Y31S, Y31H, K35R, T37A, K48E, N71R,L53I, L56I, L80I, L118I, V69A, Q74P, N88D, N88R, C125A or C125S.
 2. Themethod of claim 1, wherein the IL-2 mutein is fused or linked to a Fcpeptide.
 3. The method of claim 2, wherein the Fc peptide comprises amutation at one or more of positions of L234, L235 and G237 according tothe EU numbering system or L247, L248, and G250 according to the Kabatnumbering system.
 4. The method of claim 1, wherein the polypeptidecomprises a first chain and a second chain that form the polypeptide,wherein the first chain comprises: VH-Hc-Linker-C1, wherein VH is avariable heavy domain of the antibody that binds to MAdCAM on the cellsurface with a VL domain of the second chain; Hc is a heavy chain ofantibody comprising CH1-CH2-CH3 domain, the Linker is a glycine/serinelinker, and C1 is the IL-2 mutein fused to a Fc protein, wherein the Fcprotein is fused or linked at the N-terminus or the C-terminus of theIL-2 mutein; and the second chain comprises: VL-Lc, wherein VL is avariable light chain domain of the antibody that binds to MAdCAM on thecell surface with the VH domain of the first chain, and the Lc domain isa kappa light chain constant domain.
 5. The method of claim 1, whereinthe anti-MAdCAM antibody is a non-blocking anti-MAdCAM antibody.
 6. Themethod of claim 1, wherein the anti-MAdCAM antibody is a blockinganti-MAdCAM antibody.
 7. The method of claim 1, wherein the IL-2 muteinis an IL-2 mutein polypeptide linked to a Fc peptide, wherein the IL-2mutein comprises the amino acid sequence of SEQ ID NO: 6 with a: L80I orL118I mutation; a V69A mutation; a Q74P mutation; a N88D mutation; and aC125S mutation.
 8. The method of claim 7, wherein the Fc peptidecomprises the amino acid sequence of SEQ ID NO: 21 or SEQ ID NO:
 28. 9.The method of claim 7, wherein the IL-2 mutein is linked to a Fc peptidewith a peptide linker.
 10. The method of claim 9, wherein the peptidelinker comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 29, and SEQ IDNO:
 30. 11. The method of claim 7, wherein the Fc peptide comprises aL234A mutation, a L235A mutation, and/or a G237A mutation according tothe EU numbering system.